Method of ex vitro sowing, germination, growth and conversion of plant somatic embryos or germinants, and nutrient medium used therefor

ABSTRACT

A method of sowing a somatic plant embryo or germinant of a conifer species to facilitate subsequent development of the embryo or germinant into an autotrophic seedling. The method involves the following steps carried out ex vitro in non-sterile conditions: providing a nutrient medium comprising particles of a solid component present within a flowable or semi-solid component containing water and a carbohydrate nutrient for the embryo or germinant, dispensing a quantity of the nutrient medium onto a surface of a porous solid growth substrate for the somatic plant embryo or germinant, and contacting the plant embryo or germinant with the nutrient medium. The nutrient medium has a fluidity such that at least some of the flowable or semi-solid component containing the carbohydrate nutrient remains in contact with the embryo or germinant at least until the embryo or germinant establishes vigorous growth under environmental conditions effective for such growth. The particles of the solid component are adapted to remain in contact with the embryo or germinant after of the flowable or semisolid material dissipates, thereby providing continuing physical support for the embryo or germinant after the dissipation. The invention also relates to seedlings produced in this way and to the nutrient solution.

FIELD OF THE INVENTION

This invention relates to the sowing, germination, growth, andconversion of somatic embryos or germinants of conifer species. Moreparticularly, the invention relates to sowing, and development ofsomatic embryos or germinants ex vitro in a porous solid growthsubstrate, such as soil or peat, to allow the embryos or germinants todevelop into autotrophic seedlings and subsequently into mature plantsin non-sterile conditions.

BACKGROUND OF THE INVENTION

Dependence on the use of zygotic seed in breeding programs, wheresuccess relies on seed availability, particularly that of geneticallysuperior seeds, often leads to low returns on investment. The situationis exacerbated in tree breeding and improvement programs especiallywhere conifer species are used, because the time span from flower budinitiation to seed maturation is usually one to three years. Climaticevents and pest infection contribute to seed production variability fromyear to year (Harlow and Harrar 1968).

The development and advancement of somatic embryogenesis as a vegetativepropagation technology has made it possible to mass-produce geneticallyidentical individuals through the asexual reproduction of a sourceexplant (Tautorus et al. 1991, Roberts et al. 1995). This technologyallows for the application of clonal forestry in plantation programs.The primary advantages of clonal forestry as defined by Kleinschmit etal. (1993) and Park et al. (1998a) are:—1) the ability to capture agreater portion of the non-additive genetic gain from selectedindividuals within a breeding population; 2) the capability to rapidlyintroduce individuals with desirable traits to meet known siteconditions; and 3) the ability to carefully plan genetic diversity intoplantation programs. The primary challenge in utilising somaticembryogenesis for clonal forestry in plantation programs is thedevelopment of cost effective and scaleable methods of somatic embryoculture to produce autotrophic and acclimatised seedlings.

Somatic embryogenesis of woody plants is generally a multi-step process(reviewed by Sutton and Polonenko, 1999; U.S. Pat. Nos. 4,957,866;5,183,757; 5,294,549; 5,413,930; 5,464,769; 5,482,857; 5,506,136; thedisclosure of all of which are herein incorporated by reference). Nomatter how diverse the different somatic embryogenesis protocols mightbe, the one common step is that somatic embryos must be germinated toproduce somatic seedlings.

For zygotic embryos in natural seed, germination is supported by storednutrients within the endosperm (in angiosperms) or megagametophyte (ingymnosperms). Major forms of storage nutrients in the nutritive tissuesare starch, proteins and lipids which are broken down into simplesubstrates for us in various biochemical and physiological activitiesduring germination. For somatic embryos, nutrients needed to supportgermination must be supplied by a nutrient medium during somatic embryoculture. There are two standard approaches for germinating somaticembryos. The first approach utilises conventional in vitro methods andis generally comprised of the following steps. First, a naked somaticembryo (i.e., an embryo unprotected by any coatings) is sown, usingaseptic techniques, onto sterilised semi-solid or liquid media containedwithin a solid-support such as a Petri dish or a phytatray under sterileconditions. Second, after the somatic embryo has germinated and grownunder sterile conditions, the young seedling is transplanted intoconventional nursery growing systems. The second approach utilisesencapsulation (generally gel-encapsulation) of the somatic embryos(Carlson and Hartle 1995, Gray et al., 1995; U.S. Pat. Nos. 4,562,663;4,777,762; 4,957,866; 5,010,685; 5,183,757; 5,236,469; 5,427,593;5,451,241; 5,486,218; 5,482,857 all of which are herein incorporated byreference) prior to germination. The embryos are encapsulated in variouscoating materials to form so-called “artificial seed”, “synthetic seed”or “manufactured seed”. This encapsulation process may or may notincorporate nutrients into the encapsulating medium, and provides ameans by which the embryos can presumably be sown with conventionalnursery seeding equipment (i.e., drum seeders or fluid drill seeders)into conventional nursery growing systems. The prior art makesreferences to sowing artificial seeds ex vitro into germination mediacomprised of soil or soil-less mixes, but in fact, the prior art onlyteaches methods for germinating artificial seeds in vitro, i.e., onsterilised semi-solid laboratory media. No approaches are taught orotherwise disclosed in the prior art for sowing encapsulated somaticembryos and/or artificial seed and/or manufactured seed intoconventional growing systems using conventional sowing equipment.

The past dependence of somatic embryo germination on in vitro methodsstems from the anatomical distinction between somatic embryos andzygotic seeds: a somatic embryo lacks the nutritive tissues and theprotective seed coat that a zygotic seed possesses. Consequently,somatic embryos had to rely on exogenous nutrient supply for germinationand early growth and these events had to take place in sterileenvironments in vitro for protection against both physical andbiological damaging agents such as environmental stresses and microbialpathogens.

There are many disadvantages associated with in vitro protocols. Themost significant are: 1) the repeated manual handling of each individualembryo in the germination and transplanting steps; 2) the stringentrequirement for sterile techniques and culture conditions through allsteps until somatic germinants are transplanted out of the in vitrogermination environment into horticultural growing media; and 3) thedifficulty in acclimatizing in vitro plantlets into ex vitro nurseryenvironments. Therefore, the art of traditional in vitro protocols hasan inherent nature of low efficiency and high cost, characteristics thatare prohibitive to mass production of somatic seedlings. Theseundesirable characteristics make the commercial production of somaticseedlings less competitive than that of the zygotic seedlings (Suttonand Polonenko 1999). Automation, including robotics and machine vision,may reduce or eliminate the extensive manual-handling that is currentlynecessary to germinate naked somatic embryos. However, no commercialequipment currently exists which can reliably, aseptically, andcost-effectively perform the in vitro protocols for germination andgorwht of naked somatic embryos and subsequent transplanting ofseedlings into conventional propagation systems (Roberts et al., 1995;reviews by Sakamoto et al., 1995; and Sutton and Polonenko 1999).

There are also numerous biological and operational disadvantagesinherent in using gel-encapsulated somatic embryos. Biologically, themost significant disadvantage is the much lower germination vigour andconversion success into plants than corresponding zygotic seeds, as seenin the prior art protocols for encapsulating or otherwise coatingsomatic embryos (Redenbaugh et al., 1993; Carlson & Hartle, 1995; Grayet al., 1995). This is in sharp contrast with the germination vigour andconversion success of non-encapsulated or non-coated somatic embryos,produced with methods disclosed in the art, and then sown using aseptictechniques onto in vitro germination media in sterile conditions. Theconversion rates of germinants from in vitro sown somatic embryos canapproximate those of the corresponding zygotic seeds (e.g., greater than85%) (Gupta and Grob, 1995).

Timmins et al. (U.S. Pat. No. 5,119,588, incorporated herein byreference) recognised that “somatic embryos are too under-developed tosurvive in a natural soil environment” is and therefore must be“cultured with an energy source, such as sucrose”. They identify amethod by which plant somatic embryos can be sown into horticulturalcontainers filled with particulate soil-like substrates. Solutionscontaining compounds serving as carbon and energy sources and othernutrients, such as minerals and vitamins, are added to the substratesbefore or after the embryos are sown. Because such a “culture medium ishighly susceptible to invasion by phytopathogens, which can result indeath or retard the growth of the embryos”, they teach that thecontainers, substrate, nutrient solutions and other components of theirsystem must be biologically sterile. Somatic embryos must be sown intocontainers using aseptic techniques. Each sown container must be keptbiologically separated from the others and from the external environmentand must be kept in a sterile condition until the embryo hassuccessfully germinated and developed into a complete, independentautotrophic plant. Only after autotrophy has been reached can thesomatic seedlings be removed from the sterile conditions and thentransplanted into a conventional commercial propagation environment.Even though the art taught by such methods may be practised to producesomatic seedlings, such methods are labour-intensive and bearcharacteristics of low efficiency, high cost and impracticability formass production of somatic seedlings in a nursery environment.

The inventors named herein have previously discovered that plant somaticembryos can be directly sown ex vitro in a variety of soil or soil-likegrowing media in non-sterile conditions (PCT patent application No. WO09/965293A1, incorporated herein by reference), particularly plantsomatic embryos that have been pre-germinated (U.S. Pat. No. 6,444,467and U.S. patent application Ser. No. 09/550,110). In these patents andpatent applications, exogenous energy and nutrient sources are stilldelivered to plant somatic embryos after they have been directly sown exvitro in soil or soil-like growing media to facilitate and maximize theex vitro growth of somatic embryos.

Nevertheless, there is a constant need for improvement of thesetechniques and methods in order to overcome the disadvantages of thegermination and growth phases associated with somatic plant embryos.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method enabling theproduction of complete, independent, autotrophic plants from conifersomatic embryos preferably in conventional nursery conditions (i.e.non-sterile, ex vitro conditions).

Another object of the present invention is to provide a “nutrientmedium” to meet the requirements for successful development of conifersomatic embryos or pre-germinated conifer somatic embryos (germinants)in conventional nursery conditions.

According to one aspect of the present invention, there is provided amethod of sowing a heterotrophic somatic plant embryo or germinant of aconifer species to facilitate growth into an autotrophic seedling, whichmethod comprises the following steps: providing a nutrient mediumcomprising particles of a solid component contained within a flowable orsemi-solid component containing water and a carbohydrate nutrient forthe embryo or germinant; dispensing a quantity of the nutrient mediumonto a surface of a porous solid growth substrate for the somatic plantembryo or germinant and contacting said plant embryo or germinant withthe nutrient medium; and exposing the embryo or germinant toenvironmental conditions suitable for development into an autotrophicseedling; wherein at least the dispensing, contacting and exposing stepsare carried out ex vitro in non-sterile conditions; and wherein theparticles forming the solid component are adapted to remain in contactwith the embryo or germinant after the flowable or semi-solid componentundergoes dissipation, thereby providing continuing physical support forthe embryo or germinant after such dissipation.

The nutrient medium of the present invention can take a variety offorms, including flowable or semi-solid media of various degrees ofhardness with core and non-core ingredients. The core ingredients areusually water, simple organic and inorganic plant nutrients, andprotective agents against phytopathogens. Non-core ingredients areusually gelling and structural filling agents. Together, the core andnon-core ingredients form a water-containing flowable or semi-solidnutrient medium. The organic nutrients may comprise simplecarbohydrates, such as sucrose, glucose, fructose, and maltose as carbonand energy sources, amino acids, plant growth regulators, vitamins,fatty acids and/or other compounds that are beneficial to thegermination and growth of conifer somatic embryos. The inorganicnutrients may comprise both macro- and micro-elements that are essentialto plant life. The protective agents include pesticides such asinsecticides, fungicides, antibiotics, and/or other broad-spectrumbiocides that protect conifer somatic embryos from insect andphytopathogens that can cause death or growth retardation to conifersomatic embryos, or germinants. The gelling agents may comprise suchsubstrates as, but are not restricted to, methylcellulose, agar,agarose, phytagel, Kelcogel® and gelcarin. These gelling agents can beadded into the nutrient medium singularly or in combination. The gellingagents play a role in binding all components of the nutrient medium intoa homogeneous, lasting, nutritional matrix to support the development ofconifer somatic embryos or germinants. The solid component may comprisebiologically inert water-insoluble substrates, such as, but not limitedto, α-cellulose fiber, milled or sifted peat moss, perlite, vermiculite,clay, diatomaceous earth, coir or silica. The solid component acts as astructural filling agent which functions to anchor the conifer somaticembryo or germinant during growth while the gelling agent is dissolvingaway in the nutrient medium under the actions of soil microbes, heat,and cyclic soil wetting and drying. Preferably, the nutrient mediumshould contain a sufficient amount or proportion of the solid componentto prevent toppling (loss of intended orientation) of at least themajority (and ideally substantially all) embryos or germinants until theembryos or germinants are autotrophic and anchored in the growthsubstrate by developed roots.

The nutrient medium may be formulated in either sterile or non-sterileconditions, preferably non-sterile for reasons of simplicity. When agelling agent is not present in the nutrient medium, the nutrient mediumdoes not need to be autoclaved, or heated. Autoclaving or heating isnecessary only when there is gelling agent in the nutrient medium, asheat is needed to melt gelling agents to enable them to dissolve duringmedium preparation.

In accordance with another preferred aspect of the present invention,the nutrient medium is dispensed onto a porous solid growing substratein growing containers, preferably conventional multi-cavity miniplugnursery containers. In addition, the growing substrate should bepre-wetted and may be charged with conventional fertilizers. Thecontainers can be sterile or non-sterile, and are preferablynon-sterile. The dispensing of the nutrient medium into the containerscan be done by hand or by machinery, preferably by machinery. The amountof nutrient medium that is added into each individual container or eachindividual cavity of a multi-cavity miniplug container may varydepending upon the species of conifer somatic embryo and the containercavity size.

The conifer somatic embryos are preferably placed in direct contact withthe nutrient medium by hand or by machinery, being sown onto or into thenutrient medium so that water and nutrients can-be utilised fordevelopment of the embryo or germinant. The sowing process can bepractised in sterile or non-sterile conditions, preferably non-sterileconditions.

The sown containers can be placed in sterile or non-sterile conditions,preferably conventional non-sterile nursery conditions, to allow theplant somatic embryos or germinants to grow into complete, independent,autotrophic plants. Conventional nursery conditions typically providehigh relative humidity, warm air temperature, and sufficient light tomeet the growth and development requirements of germinating orpre-germinated conifer somatic embryos. Consequently, the somaticseedlings produced from somatic embryos will have not required thetransplanting or acclimation measures commonly associated with in vitroseedlings.

If desired, the somatic plant embryo may be pre-germinating prior tocontacting the somatic plant embryo with the nutrient medium. Theembryos are preferably sown with their root radicles pointing down andthe remainder of the embryo in an upright position.

The flowable or semi-solid component is preferably a material having astructural integrity such that at least some of said flowable orsemi-solid component containing the carbohydrate nutrient remains incontact with said embryo or germinant until said embryo or germinant iscapable of autotrophic growth under the environmental conditions and afluidity under said environmental conditions such that it may bedispensed under gravity or pressure from an orifice onto the poroussolid growth substrate. The somatic plant embryo or germinant may becontacted with the nutrient medium after the nutrient medium has beendispensed onto the surface of the porous growth substrate, before thenutrient medium has been dispensed onto the surface of the porous growthsubstrate, or as said nutrient medium is dispensed onto said surface ofthe porous growth substrate. Most preferably, at least a part of theembryo or germinant remains exposed to the air after being sown incontact with the nutrient medium. This allows oxygen exchange with theembryo or germinant during its subsequent growth.

The porous solid growth substrate is preferably in the form of a bodyprovided with a depression formed in the upper surface, and the nutrientmedium is dispensed into the depression onto the surface. The embryo isthen at least partially inserted into the depression in contact with thenutrient medium. The embryo is preferably sown naked.

The solid component of the nutrient medium may be of any solid,water-insoluble, biologically inert material or soil-like substrate suchas α-cellulose fiber, milled or sifted peat moss, perlite, vermiculite,clay, diatomaceous earth, coir or silica. The solid components mayinclude equi-axial particles or elongated particles, such as flexiblefibers like those of α-cellullose and milled or sifted peat moss. Thesolid material is preferably present in the range up to 10% w/v, andmore preferably between 3 and 8% (w/v), more preferably between 3 and 5%(w/v). The medium preferably also contains methylcellulose to preventseparation of the medium during storage. The concentration of themethylcellulose in the nutrient medium is between 0 and 6% w/v, morepreferably 0.3 to 2% w/v. The nutrient medium is preferably a flowableor semi-solid medium containing the carbohydrate in dissolved form. Ifthe nutrient medium contains a semi-solid component, it is preferably aflowable gel containing the carbohydrate in dissolved form. The gel maybe formed by using a gelling agent, e.g. methylcellulose, agar, agarose,phytagel, gellan gum (e.g. Kelcogel®), and gelcarin. When the gellingagent is agar, it is preferably present in a concentration in thenutrient medium of less than 1.2% w/v, preferably 0.3 to 0.8% w/v. Whenthe gelling agent is phytagel, it is preferably present in aconcentration in the nutrient medium between 0 and 1.2% w/v, preferably0.1 to 0.4% w/v. When the gelling agent is gelcarin, it is preferablypresent in a concentration in the nutrient medium between 0 and 1.2%w/v, more preferably 0.3 to 0.8% w/v.

The nutrient medium may additionally contain one or more mineralcompounds, vitamins, amino acids, plant growth regulators and pestcontrol compounds.

The carbohydrate nutrient present in the nutrient medium may be amonosaccharide, such as glucose or fructose, or an oligosaccharide, suchas sucrose, maltose, raffinose or stachyose. The carbohydrate source inthe nutrient medium may also be a combination of monosaccharides, acombination of oligosaccharides, or a combination of monosaccharideswith oligosaccharides. Ideally, the carbohydrate nutrient is sucrosepresent in the nutrient medium in an amount in the range of in the range1 to 10% (w/v), preferably 1 to 6% (w/v), and more preferably 1 to 4%(w/v). Alternatively, the carbohydrate nutrient may be maltose presentin the nutrient solution in an amount in the range of in the range of 1to 10% (w/v), preferably 1 to 6% (w/v), more preferably 1 to 4% (w/v).

The embryos or germinants may be of any conifer species, e.g. embryosare from the family Pinaceae, preferably of the pine (Pinus) genusincluding a variety of pine species and hybrids, e.g. Pinus hybridsselected from the group consisting of Pinus rigida×Pinus taeda, Pinustaeda×Pinus rigida, Pinus serotina×Pinus taeda, and Pinus taeda×Pinusserotina. Particularly suitable are embryos of loblolly pine (Pinustaeda L.), radiata pine (Pinus radiata D. Don.). Also suitable areembryos of the spruce (Picea) genus including a variety of sprucespecies and hybrids, as well as embryos of the species Douglas-fir(Pseudotsuga menziesii Mirb. Franco).

The somatic embryos are generally mature embryos in at least one of thefollowing states:

-   -   a) freshly matured embryos, having received no desiccation        treatments after maturation and prior to being sown in direct        contact with said nutrient medium;    -   b) partially dried by a high relative humidity drying treatment        after maturation and prior to being sown in direct contact with        said nutrient medium;    -   c) desiccated by a severe drying treatment after maturation and        prior to being sown in direct contact with said nutrient medium;    -   d) cold-stored at temperatures between 10 and 0° C. after        maturation and prior to being sown in direct contact with said        nutrient medium; and    -   e) frozen-stored at temperatures between 0 and −190° C. after        maturation and prior to being sown in direct contact with said        nutrient medium.

The somatic embryos, before being sown in direct contact with thenutrient medium, may have received at least one of the followingpre-treatments:

-   -   (a) in an original state without a pre-germination treatment;    -   (b) the embryos are pre-germinated.

The embryos are preferably sown in a multiple cavity miniplug tray usingsoil, or a soil-like material as the substrate. Examples of soil-likematerials are peat and peat products, which may be used in combinationwith an extender such as vermiculite or perlite. However, non-peat basedsoil-like materials may also be used. The soil-like material may bebound together with a polymer.

The non-sterile growing conditions used for germinating and growing theembryos may include temperatures within the range of 10 and 35° C., morepreferably 15 to 30° C., atmospheric relative humidity within the rangeof 20 and 100%, more preferably 60 to 100%, carbon dioxide concentrationwithin the range of 0.003 and 3%, preferably between 0.003 and 0.03%;light level within the range of 0 and 2000 μmol m⁻²s⁻¹Photosynthetically Available Radiation (PAR), in a light/dark diurnalcycle within the range between 24 h/0 h and 0 h/24 h. The light/darkdiurnal cycle is preferably between 16 h/8 h and 20 h/4 h, and the lightlevel is preferably between 15 and 500 μmol m⁻²s⁻¹ PAR. Ideally, thetemperature, atmospheric relative humidity, carbon dioxide, light, anddiurnal light/dark cycle conditions are created in non-sterile growingspaces such as conventional greenhouses, specifically constructed growthchambers, and/or specifically constructed growth rooms.

Additional nutrient medium may be supplied after sowing the embryos orgerminants, e.g. a carbohydrate-containing and/or macro or micronutrient-containing aqueous liquid applied by spraying, misting,drenching or irrigating. Spraying and misting are preferred in order toreduce the speed at which the original nutrient medium is dispersed.

According to another aspect of the invention, there is provided a methodof growing an autotrophic seedling from a somatic plant embryo orgerminant of a conifer species, which method comprises the followingsteps carried out ex vitro in non-sterile conditions: providing anutrient medium comprising particles of a solid component present withinan aqueous, flowable or semi-solid component containing a carbohydratenutrient for the embryo or germinant; dispensing a quantity of thenutrient medium onto a surface of a porous solid growth substrate forthe somatic plant embryo or germinant held in a container; contactingthe plant embryo or germinant with the nutrient medium; and subjectingthe embryo to environmental conditions to cause development of theembryo or germinant into an autotrophic conifer seedling; wherein thenutrient medium has a cohesiveness such that at least some of theflowable or semi-solid component containing the carbohydrate nutrientremains in contact with the embryo or germinant as the embryo orgerminant proceeds to full germination and/or conversion to autotrophyunder appropriate environmental conditions; and wherein the particles ofthe solid component are adapted to remain in contact with the embryo orgerminant after of the flowable or semi-solid material dissipates,thereby providing continuing physical support for the embryo orgerminant after such dissipation.

The present invention also relates to the nutrient medium used in themethod above, and to seedlings produced by the method.

In preferred forms of the invention, the method of sowing or growingsomatic embryos or germinants begins with the preparation of conifersomatic embryos prior to germination. This can be achieved by methodsalready known in the art.

The entire process of nutrient medium preparation, nutrient mediumapplication, embryo or germinant sowing, germination, and growth can be(and preferably is) practised in non-sterile conditions. Each of thesesteps can be automated if so desired.

Consequently, aseptic techniques, equipment, and conditions are nolonger necessary for successful development of conifer somatic embryosor germinants to produce autotrophic seedlings.

There are several advantages associated with the present invention, atleast in its preferred forms. One advantage is that the nutrient mediumprovides a continuous supply of water and nutrients to the conifersomatic embryo or germinant at least for early ex vitro growth anddevelopment until the flowable or semi-solid component dissipates. Waterin the nutrient medium also buffers the conifer somatic embryo orgerminant from water stress in open ex vitro conditions, which couldarise from low air humidity or low soil water content.

Another advantage, at least of preferred forms of the invention, is thatthe nutrient medium contains a solid component that provides continuingphysical support to the is embryos or germinants as the other componentsdrain away or disappear, thus allowing the embryos or germinants toremain in a proper upright position until a root develops to anchors itinto the porous solid growth substrate.

Another advantage of this invention, at least in its preferred forms, isthat the nutrient medium, by virtue of its flowable or semi-solidproperties, provides a homogenous matrix for close contact between theconifer somatic embryo or germinant and the matrix. This facilitates theabsorption of nutrients and water by the conifer somatic embryos orgerminants and eliminates poor plant-soil contact that is oftenassociated with ex vitro seedlings on non-semi-solid or non-flowable,particulate-like growing media.

In its preferred forms, another advantage of this invention is that theconifer somatic embryos or germinants may be sown directly into soil (orother solid growth substrate) in nursery containers and remain there forsubsequent growth stages. Consequently, there is no need for costlytransplanting and acclimation of somatic seedlings, which aredisadvantageous features of in vitro seedlings.

An additional advantage of this invention, at least in preferred forms,is that the ex vitro conifer somatic embryos or germinants do notrequire aseptic techniques, equipment or conditions for development. Thesowing and transplanting into large containers, when needed, can be doneusing automated nursery equipment. This allows for low-cost massproduction of high quality somatic seedlings.

If desired, the nutrient medium may be added into a solid growthsubstrate in containers. Solid growth substrates include, but are notlimited to, soil or soil-like substrates, natural or artificial.Consequently, conifer somatic embryos or germinants will grow directlyinto soil without the need of multi-step hand or machine-handling. Thesoil or soil-like substrates can be sterile or non-sterile, but arepreferably non-sterile.

The solid growth substrate may be provided in conventional nurserycontainers. Such containers can vary in size, but are preferablymulti-cavity miniplug containers. The containers can be sterile ornon-sterile, but are preferably non-sterile.

The conifer somatic embryos or germinants may be sown directly onto orinto the nutrient medium in the solid growing support in the containers.Such a sowing process can be performed either by hand or by machinery,but preferably by machinery.

The entire process may be operated in non-sterile conditions, from thesteps of nutrient medium formulation, nutrient medium application,embryo or germinant sowing, and development of the conifer somaticembryos or germinants into complete, independent, autotrophic plants insoil. Consequently, no aseptic techniques, equipment, conditions, and noacclimation steps as commonly required by in vitro germinants areneeded. Seedlings so produced may be grown in the original container ormay be transplanted at a later date into large or commercial sizedcontainers.

The non-sterile conditions may be those found in a conventional nurseryenvironment. Therefore, the entire process can be practised inaccordance with the operation of a nursery.

The present invention includes the above objects and features takenalone or in combination. These and other features, objects, andadvantages of the present invention will become readily apparent tothose skilled in the art upon reading the following detaileddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1 to 5 schematically show examples of steps involved in thesowing, development and growth of somatic embryos in according to apreferred method according to the present invention; and

FIGS. 6 and 7 are graphs showing the results of tests carried out in theExamples below.

DEFINITIONS

A number of terms are known to vary in meaning in the literaturedescribing this art. The following definitions are believed to be thosemost commonly used in the fields of botany and plant somaticembryogenesis, and are consistent with the usage of the terms in thepresent specification, including the claims. These definitions willassist in the understanding of the detailed description of the presentinvention that will follow.

“Acclimatise” or “acclimatize” refers to the adaptation to a newtemperature, climate, or environmental condition, etc. For embryosgerminated in vitro on semi-solid media, acclimatisation refers to theprocesses involved in the seedlings successfully adapting to ex vitroenvironments in the greenhouse or nursery, and subsequently resumingvigorous growth and development.

“Ampicillin” is an antibiotic.

“Autotrophic” refers to the stage of plant development in which thephotosynthetic organelles, related enzymes and biochemical pathways arecompletely functional and capable of converting light energy,atmospheric carbon dioxide and water into the prerequisite carbohydrates(e.g., glucose) necessary to sustain further plant growth anddevelopment. Autotrophic plants are able to survive and grow undernormal soil conditions.

“BA” is benzyl adenine, a cytokinin-type of plant growth regulator. Themain physiological effect of BA is to stimulate cell division.

“Benlate” is a registered trademark for benomyl (methyl1-(butylcarbamoyl)-2-benzimidazolecarbamate), which is a fungicide.

“Clone”, when used in the context of plant propagation, refers to acollection of individuals having the same genetic constitution and isproduced from an individual plant or a culture that arises from anindividual explant.

“Conversion” refers to the transition from a heterotrophic stage ofplant development to an autotrophic stage of plant development. The term“converted” as applied to an embryo or germinant means an embryo orgerminant that has undergone conversion and is autotrophic.

“Desiccation” refers to the drying of a somatic embryo by any means to awater content less than that of the original hydrated embryo.Desiccation processes may include (a) mild desiccation, whichencompasses water contents in the 36-55% water content range, and (b)severe desiccation, which occurs at water contents less than 36%,usually in the range of 5-30%. A fully desiccated viable embryo is ableto survive freezing, and after rehydration, is able to successfullycomplete the germination process and convert to a normal, autotrophicplant.

“Endosperm” is strictly an angiosperm seed structure, and is alwaystriploid due to double fertilisation. The endosperm contains thenutritive reserves required for zygotic embryo development and growthwhen the germination process is started.

“Explant” is the organ, tissue or cells derived from a plant andcultured in vitro for the purposes of starting a plant cell or tissueculture.

“Flowable” means that a material flows under its own weight or can becaused to flow under light force or pressure (i.e. forces or pressure ofthe kind encountered in sowing operations).

“Frozen storage” refers to storage of embryos at less than the freezingpoint of water, and preferably at a temperature in the range of −10° C.to −190° C. “GA” is an abbreviation for gibberellins, a group of relatedgrowth regulator isomers (e.g., GA₃, GA₄, and GA₇) that are naturallysynthesized as a normal part of plant metabolism. The main physiologicaleffect of GA is to stimulate elongation of individual plant cells.Exogenous applications of GA can be used to stimulate, manipulate andaccelerate the initiation and growth of shoots and roots.

“Gelcarins” are purified carrageenans, which are a naturally occurringfamily of polysaccharides derived from red seaweed.

“Genotype” refers to the genetic constitution of an organism, acquiredfrom its parents and available for transmission to its offspring. Whenused in the context of asexual plant propagation, genotype isinterchangeable for clone.

“Germinant” refers to a somatic embryo that has been pre-germinated butnot converted. “Gnatrol®” is a biological larvicide. The activeingredient of this product is Bacillus thuringiensis subspeciesisraelensis.

“Heterotrophic” refers to the stage of plant development when thephotosynthetic organelles, related enzymes and biochemical pathways arestill not completely functional or capable of converting light energy,atmospheric carbon dioxide and water into the prerequisite carbohydrates(e.g., glucose) necessary to sustain further plant growth anddevelopment. Consequently, heterotrophic plants still require anexogenous supply of carbon and energy resources in the growth mediumsuch as sucrose, to sustain normal growth and development until theplants become completely autotrophic. By definition, heterotrophicplants are not able to survive and grow under normal soil conditions.

“HRHT” stands for a high relative humidity drying treatment, which is apartial drying process for somatic embryos performed in a high relativehumidity range of 85-99.9%. The treatment is carried out by the methodof the Roberts patent (U.S. Pat. No. 5,183,757, incorporated herein byreference).

“IAA” is indole-3-acetic acid, an auxin-type plant growth regulatornaturally synthesised as a normal part of plant metabolism. The mainphysiological effect of IAA is to stimulate meristematic cell division,cell enlargement and differentiation. Exogenous applications of IAA canbe used to stimulate, manipulate and accelerate the initiation andgrowth of shoots and roots.

“IBA” is indole-3-butyric acid, a synthesised analogue of IAA. IBA canbe used to affect the initiation of roots and shoots in the same manneras exogenous applications of IAA.

“Megagametophyte” refers to haploid nutritive tissues of gymnospermseed, maternal in origin within which the gymnosperm zygotic embryosdevelop.

“Nutrients” are the inorganic micro- and macro-minerals, vitamins,hormones, organic supplements, and carbohydrates necessary for culturegrowth and somatic embryo development.

“Nutrient solution” refers to water containing a dissolved nutrient ormixture of nutrients.

“Penicillin” is an antibiotic.

“Pre-germination” refers to a process-of contacting a mature somaticembryo with medium for any period of time until onset of autotrophicdevelopment.

“Root radicle” refers to the meristematic end of a germinating embryofrom which roots develop. This is an area in which undifferentiated celldivision is rapid, with the resulting cells then undergoing a period ofelongation behind the root radicle before transformation intodifferentiated cells.

“Seed” refers to the ripened ovule consisting of the zygotic embryo, itsproper nutritive tissues and coat.

“Seedling” refers to a conifer somatic plant that is autotrophic and/orhas a well-developed epicotyl.

“Semi-solid” used in connection with a component of a nutrient mediumrefers to a consistency of the component of the nutrient medium suchthat it does not flow under its own weight, but can be made to flow byapplying light pressure or force.

“Somatic embryo” refers to a plant embryo formed in vitro fromvegetative (somatic) cells by mitotic division of cells. Early stagesomatic embryos are morphologically similar to immature zygotic embryos,and comprise a region of embryonic cells subtended by elongatedsuspensor cells.

“Somatic embryogenesis” is the process of initiation and development ofembryos in vitro from somatic cells and tissues.

“Water potential” is defined as the free energy per unit volume ofwater, assuming the potential of pure water to be zero under standardconditions. Water potential of a cell or plant is the sum of osmotic,turgor, and matric potential. Osmotic potential is the componentproduced by solutes dissolved in the cell sap. Turgor potential isproduced by diffusion of water into protoplasts enclosed in cell wallsthat resist expansion. Matric potential refers to water held bycolloids, on surfaces and in microcapillaries in the cells and cellwalls. Both the osmotic and matrix potential reduce the free energy ofwater whereas turgor potential increases the free energy of water. Waterpotential in a cell or plant is usually negative. Water potential andits components are customarily expressed in pressure units, such asPascal (Pa) or mega-Pascal (MPa).

“Water stress” refers to situations in which plant water potential andturgor potential are reduced enough to interfere with normal functioningunder dehydration (drought) conditions. Water stress can also be causedby excessive water in the rooting zone.

“Zygotic embryo” is an embryo derived from the sexual fusion of gameticcells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of sowing a somatic plantembryo or germinant, nutrient media for use in the method, and a methodfor producing complete, independent, autotrophic seedlings utilizing themethod of sowing. In particular, the invention is concerned with somaticembryos or germinants from conifer species and methods carried out innon-sterile ex vitro conditions, preferably using essentially nakedembryos or germinants. This enables the invention, at least in itspreferred forms, to be carried out relatively inexpensively, on a large(even mass-produced) scale of operation, using equipment and facilitiesthat are common in the plant-growing concerns.

The invention makes use of a carbohydrate- and water-containing nutrientmedium that is provided in contact with the somatic embryos or germinantas they are sown on or in a solid porous growth substrate. The nutrientmedium provides for delivery of water and nutrients (including one ormore carbohydrates) to conifer somatic embryos or germinant for growth,and also preferably provides physical support for the embryo to keep itoriented in the correct (upright) position until sufficient root growthfor anchoring the plant has taken place. The nutrient medium contains acomponent consisting of solid particles and a component that is flowableor semi-solid. The fluidity (viscosity) of the medium is preferably suchthat the flowable or semi-solid component is not immediately absorbedinto a porous solid growth substrate on which the medium is dispensedduring sowing. In fact, the fluidity is such that the medium, and thecarbohydrate it contains, remains in contact with the embryo during itsperiod of germination. The medium can also provide immediate physicalsupport for the embryo. When the flowable or semi-solid component iseventually fully absorbed by the growth material or otherwise removedfrom the surroundings of the embryo, the solid component (solidparticles) remains to provide continued physical support for the embryoso that it can continue to develop roots and to mature withoutdisruption caused by a lack of physical support. The method, includingthe manufacturing and application of the nutrient medium, can bepractised in conventional non-sterile conditions using conventionalhorticultural practices, equipment, and facilities.

In a particularly preferred form, the following steps may be carriedout:

-   -   1. The conifer somatic embryos are pre-germinated. The        pre-germination treatment may or may not include initial culture        on a semi-solid medium to be followed by liquid culture. Initial        culture on a semi-solid medium may not be essential particularly        if: (a) fresh, mature conifer somatic embryos are to be used;        and (b) conifer somatic embryos are not to be pre-germinated.        After the completion of the pre-germination treatment, the        vigour and synchrony of the pre-germinated conifer somatic        embryos are generally substantially increased as compared to        those conifer somatic embryos that are not pre-germinated. The        art of the pre-germination treatment is detailed in U.S. Pat.        No. 6,444,467 and U.S. patent application Ser. No. 09/555,110        (incorporated herein by reference).

The liquid culture step of the pre-germination methods starts with thetransfer of plant somatic embryos into a vessel containing a liquidmedium. The liquid medium contains at least one source of carbohydrates,such as, but is not restricted to, sucrose in the range of 1-6% (w/v).The liquid medium also contains other types of nutrients that mayfurther facilitate the various biochemical and physiological processesoccurring during development. Such nutrients include, but are notrestricted to, inorganic mineral elements, amino acids, vitamins, andplant hormones.

The conifer somatic embryos are preferably cultured in liquid medium fora period of time in the range of one to five days and preferably betweenthree to four days. Throughout the liquid culture, the embryos aresuspended within the solution and are kept in constant motion. Anon-limiting example of how this might be accomplished is by securingthe vessels containing the embryos and liquid medium onto a shaker tablewhich is revolving at a rate in the range of 40 -120 rpm, preferably inthe range of 80-100 rpm. Since it is known in the art that zygotic seedsof certain plant species will germinate in the dark while zygotic seedsof other plant species require light for successful germination,pre-germination may be practised in either the presence or absence oflight.

It is preferable throughout pre-germination that aseptic techniques beused. It is therefore preferable that the pre-germination vessels andthe media used during pre-germination are sterilised prior to theaddition of the conifer somatic embryos, and that an aseptic techniqueis used when adding embryos to media. It is also preferable that thecontents of the pre-germination vessels be maintained in a sterilecondition during the pre-germination process. However, it should benoted that is not essential to pre-germinate conifer somatic embryos inorder to practise this invention.

-   -   2. The conifer somatic embryos (either pre-germinated or not        pre-germinated) are sown by hand or by machinery, preferably by        machinery, into nursery containers containing a porous solid        horticultural growing substrate (actually, such substrates        normally contain three phases all present simultaneously, i.e.        solid particles, moisture and air—but would normally be referred        to as solid substrates) and a flowable or semi-solid nutrient        medium. The nutrient medium contains at least one source of        carbohydrate that is a nutrient for the embryo. Although the        preferred carbohydrate is sucrose, preferably present in the        medium in an amount in the range of 1-6% (w/v), this invention        can be practised with other carbohydrates such as fructose,        glucose, maltose, galactose, mannose, lactose, etc. Furthermore,        the nutrient medium may contain, if so desired, a mixture of two        or more carbohydrates. If mixtures of carbohydrates are used in        the nutrient medium, then the appropriate concentrations of each        carbohydrate should be experimentally determined in advance by        the use of rate-selection studies known to those skilled in this        art.

In addition to carbohydrates, the nutrient medium may also contain, ifso desired, other types of nutrients that may further facilitate variousbiochemical and physiological processes occurring during developmentalphases from germination to conversion. Such nutrients include, but arenot restricted to, inorganic mineral elements, organic acids, vitaminsand plant hormones. The following is a non-limiting example of thispractise. The nutrient medium may contain sucrose in the range of 1-6%(w/v), and a mixture of mineral nutrients formulated to deliver: 350 mgnitrogen/l, 186 mg phosphorus/l, 469 mg potassium/l, 25 mg calcium/l,19.5 mg magnesium/l, 364 mg sulphur/l, 171 mg chlorine/l, 3 mgmanganese/l, 0.5 mg zinc/l, 3 mg iron/l, 0.01 mg iodine/l, 0.6 mgboron/l, 0.01 mg molybdenum/l, 0.01 mg cobalt/l, and 0.01 mg copper/l,1.2 mg L-glutamine/l, 0.1 mg L-alanine/l, 0.04 mg L-cysteine-HCl/l, 1.2mg L-arginine/l, 0.02 mg L-leucine/l, 4.0 mg glycine/l, 4.0 mgL-serine/l, 1.0 mg L-asparagine/l, and 1.0 mg L-proline/l. Furthermore,if so desired, IBA, a plant growth regulator, may be added alone at aconcentration of 0.05˜0.1 μmol/l or in combination with one or both ofGA and BA, each at a concentration of 0.05˜0.1 μmol/l.

Furthermore, if so desired, pest control products such as antibiotics orfungicides may be added to the nutrient medium. A non-limiting exampleis the addition of Benlate (0.1 g/l) and/or Ampicillin (0.1 g/l).

Furthermore, if so desired, gelling agents may be added to the nutrientmedium either singly or in combination. The gelling agent binds allcomponents of the nutrient medium to form a flowable or semi-solid,nutritious matrix for development of plant somatic embryos orgerminants. A non-limiting example is the addition of phytagel (0.2-0.3%w/v), agar (0.3-0.4% w/v), gelcarin (0.3-0.4% w/v) or methyl-cellulose(0.4-1.0% w/v).

Furthermore, a particulate solid component is incorporated into thenutrient medium, e.g. a filling agent such as α-cellulose, milled orsifted peat moss, perlite, vermiculite, clay, diatomaceous earth, coiror silica. The structural filling agent (solid component) supports theconifer somatic embryo germinant as the flowable or semi-solid dissolvesaway under the action of various forces such as soil microbes, heat,wetting and drying. A non-limiting example is α-cellulose or milledpeatmoss in the range of 3 - 6% w/v. The particulate solid may be in theform or regular or irregular generally equi-axial particles or clumps,or may be in the form of elongated fibers, with elongated fibers beingmost preferred. The solid should be such that it remains around oragainst the embryo after the remainder of the nutrient medium hasdispersed and provides a matrix or body that supports the embryo againsttoppling over or displacement.

It has also been found preferable to incorporate methylcellulose intothe nutrient medium. This helps to prevent separation of the medium intosolid and liquid phases, and it prevents clumping of the medium, andalso helps to retain the medium on the solid porous substrate withoutrapid uptake by the substrate. It has been found that the addition ofabout 0.75% -1.0% by wt. methylcellulose in combination with 4-5% w/vα-cellulose or milled or sifted peat moss improves the consistency ofthe medium.

The nutrient medium is prepared prior to being added onto a porous solidhorticultural growing substrate. It is not necessary to autoclave orheat the nutrient medium unless a gelling agent is included. If heatingis required, the nutrient medium solution should be autoclaved or heatedfor a period of time in the range of 2 to 25 minutes, preferably 3 to 20minutes, most preferably 3 to 10 minutes. Autoclaving or heating may bedone using various forms of equipment, such as, but not restricted to,an autoclave, hot plate, stove, oven, or microwave oven. The autoclavedor heated nutrient medium can be added to the three-phase conventionalgrowing substrate while it is still hot, but preferably it is cooled toroom temperature. When a gelling agent is included at a low percentageconcentration, such as phytagel at 0.2% w/v, agar at 0.3% w/v, orgelcarin at 0.3% w/v, the nutrient medium is a soft semi-solid aftercooling. It can be agitated into a viscous but flowable medium. Whensuch gelling agents are included at a lower percentage concentrationthan indicated above, the nutrient medium is viscous but flowable aftercooling. In its preferred form, the nutrient medium should be kept fluidfor dispensing onto the growing substrate.

Various devices with precision volume control mechanisms may be used todispense the nutrient medium onto the growing substrate. A non-limitingexample is a repeat pipette, a multi-channel pipette, or a precisionpneumatic dispensing pump. In preferred forms of the invention, thegrowing substrate is pre-wetted with water and/or charged withfertilizer at 50 ppm nitrogen (such as 11-41-8,nitrogen-phosphorus-potassium) as well as with micro-nutrients.Generally, the nutrient medium is dispensed first onto the surface ofthe solid substrate, or in a depression formed in the surface, to form apool, and then an embryo or germinant is placed at least partially inthe pool of nutrient medium, ensuring that the embryo is orientedcorrectly for subsequent germination and growth. However, it would bepossible to sow the embryo or germinant first, and then to dispense thenutrient medium to surround the embryo, at least partially. It isfurther contemplated that the embryo and nutrient medium may bedispensed at the same time, i.e. the embryo would be suspended in thenutrient medium, and the embryo would be dispensed within a droplet orpool of the nutrient medium onto the surface of the solid substrate orinto a depression formed in the surface.

It is apparent from the above description that this entire step can benon-sterile. Consequently, commencing from this step, aseptic techniqueand sterile conditions are not required to successfully practise thisinvention.

-   -   3. Nursery containers sown with conifer somatic embryos or        germinants are placed in conventional plant propagation        environments (such as chambers, growth rooms or conventional        greenhouses) in which light, temperature, atmospheric humidity,        carbon dioxide, and water content of the rooting substrate can        be controlled. This controlled nursery environment facilitates        the further development of conifer somatic embryos or germinants        into seedlings. The following conditions provide a non-limiting        plant propagation environment for conifer species such as white        spruce, loblolly pine, radiata pine, and Douglas-fir: light at        20-300 μmol m⁻²s⁻¹ Photosynthetically Available Radiation (PAR),        18 hour day/6 hour dark photoperiod, 20-26° C. day and night air        temperatures, 80-100% relative humidity for the first week with        a gradually decrease in humidity to 50-70% over a three week        period, and 0.015% carbon dioxide concentration. The ambient        light intensity and diurnal photoperiod, temperature,        atmospheric humidity and other such factors may be adjusted as        required for each specific species during somatic embryo or        germinant growth and their subsequent development and conversion        into completely functional seedlings.    -   4. During the growing period, if so desired, an aerosol in the        form of a mist or spray may be supplied to the surface of the        nursery containers sown with somatic embryos or germinants. The        aerosol may contain the necessary carbohydrate and other        nutrient. Alternatively, the supplemental nutrients may be        applied to the horticultural growing substrate in liquid form,        directly adjacent to the plant somatic embryos, with a device        having a precision volume control mechanism such as a pipette.        However, it is not essential that carbohydrates, amino acids,        vitamins, and plant hormones be supplemented during the ex vitro        development processes. It is, however, necessary to continue the        supplementation of macro- and micro-mineral elements during        growth and development of the conifer somatic embryos or        germinants to ensure successful autotrophic development. These        macro- and micro-mineral elements may be supplemented through        incorporation of commercial fertilisers in the normal greenhouse        growing practices.    -   5. Conifer seedlings so produced may continue to grow to        commercial size in the original containers under normal        greenhouse growing regimes if the container sizes are        sufficiently large to meet the growth requirements.        Alternatively, seedlings grown in “miniplug” containers may be        transplanted at a later date into large-sized containers to        continue their growth and development under normal greenhouse        conditions. Or, seedlings grown in miniplug containers may be        transplanted at a later date into bare-root nursery beds if so        required.

A feature of the present invention, at least in its preferred forms, isthat the water and nutrient requirements of conifer somatic embryos orgerminants for early growth and development are met through the presenceof a nutrient medium prior to the plants becoming completelyautotrophic. The addition of a nutrient medium allows the conifersomatic embryos or germinants to grow in a normal nursery environment.Because, at least in its preferred forms, the nutrients are contained ina gelled nutrient medium, the nutrients are long lasting. Also, becausefrequent irrigation of the pre-wetted and/or fertilized growingsubstrate is not required (though infrequent light misting can bebeneficial), nutrients in the nutrient medium are not subjected toleakage from the growing substrate. Consequently, all nutrientsoriginally incorporated in the nutrient medium and intended for theembryos or germinants can be utilised by the embryos or germinants forgrowth. Further to this, because conifer somatic embryos or germinantsare directly sown on or in the nutrient medium, the nutrients in thenutrient medium are readily available. The advantage of this feature isthat the presence of the nutrient medium reduces or eliminates the needfor repeated application of exogenous nutrients after the conifersomatic embryos are sown, as described in PCT patent application no. WO09/965293 A1, and U.S. Pat. No. 6,444,467 (incorporated herein byreference).

Another feature of the present invention, at least in its preferredforms, is that the formulation of the nutrient medium can vary accordingto the requirements of somatic embryos or germinants of different plantspecies. The requirements of different species may be experimentallydetermined in advance by studies. The design and performance of suchstudies are known to those skilled in the art. Furthermore, the nutrientmedium may contain essential nutrients, but may also contain othercomponents that are deemed beneficial for development of plant somaticembryos or germinants.

Another feature of the present invention, at least in its preferredform, is that although there exists a nutrient medium in the growingsubstrate, it does not exclude, if so desired, the addition of extraexogenous nutrients. These exogenous nutrients can include, but are notrestricted to, carbohydrates and minerals applied by either aerosols orby injection or drenching. The nutrient solutions can be applied with,but are not restricted to, conventional misting and/or foggingequipment. The nutrients can be applied individually or combined intoone solution. A non-limiting example of this practise is the applicationof a nutrient solution containing 3% sucrose (w/v), 350 mg nitrogen/l,186 mg phosphorus/l, 469 mg potassium/l, 25 mg calcium/l, 19.5 mgmagnesium/l, 364 mg sulphur/l, 171 mg chlorine/l, 3 mg manganese/l, 0.5mg zinc/l, 3 mg iron/l, 0.01 mg iodine/l, 0.6 mg boron/l, 0.01 mgmolybdenum/l, 0.01 mg cobalt/l, and 0.01 mg copper/l. If so desired, theplant growth regulator IBA may be added alone at a concentration of 0.1μmol/l or in combination with GA and BA, separately or together, each ata concentration of 0.05-0.1 μmol/l. Another non-limiting example is toseparately apply sucrose and mineral elements. The mineral nutrients canbe supplied as a commercial formulation such as but not restricted to10-52-10 (nitrogen-phosphate-potassium) or 11-41-8(nitrogen-phosphate-potassium). Another alternative non-limiting meansof supplying exogenous nutrients to somatic embryos or germinants sownonto growing substrate within nursery containers is to irrigate or“drench” the growing substrate with nutrient solutions formulated aspreviously described by using any conventional or specially designedmultiple mini-injectors. Alternatively, the exogenous nutrients may bedelivered to each individual embryo or germinant with a device having aprecision Volume control mechanism, such as but not restricted to, arepeater pipette, a multi-channel pipette or pneumatic pump.

Another feature of the present invention, at least in its preferredforms, is that special hygienic and/or aseptic and/or sterile handlingmethods and/or equipment and/or facilities are not required tosuccessfully handle, sow and grow desiccated or hydrated fresh embryosor pre-germinated conifer somatic embryos. All steps, excludingpregermination, may be carried out in non-sterile, non-hygienic and/ornon-aseptic conditions, i.e., those types of conditions typicallyencountered in conventional horticultural or forest nursery plantpropagation environments.

Still another feature of the present invention, at least in itspreferred forms, is that the sowing and propagation of conifer somaticembryos or germinants can be practised with a wide variety ofnon-sterilised growing substrates in a wide variety of non-sterilisedgrowing containers commonly used in conventional plant propagation. Thepreferred growing substrate is peat-based and has been formulatedspecifically for germination of zygotic seed. Examples of such mixturesare (a) 100% short-fibre peat product polymerised with a water-bindingpolymer, (b) 15.2 cu. ft of peat, 8 cu. ft. of vermiculite, 680 grams ofdolomite lime, and 300 grams of Micromax®, and (c) 16.2 cu. ft. of peat,6.75 cu. ft. perlite, 4 cu. ft. vermiculite, 6 kilograms of dolomitelime, 1.5 kilograms of gypsum, 375 grams of potassium phosphate, 250grams Micromax®, and 35 grams of wetting agent. Alternatively,commercially formulated mixes can also be used with the presentinvention. It is preferred that the peat-based growing substrate bemoistened to a water content in the range of 59-75% and then dispensedinto growing containers. It should be noted that the present inventioncould be practised in substrates other than peat-based mixtures. Suchsubstrates could be, but are not restricted to, the following materials:peat plugs, composted or shredded coconut husk fibres commonly referredto as “cor” or “coir”, extruded foams, and rock wool. Regardless of therooting substrate chosen, its physical characteristics should enabledevelopment and maintenance of a high relative humidity in the gaseousphase, i.e., in excess of 75% RH within the substrate, while minimisingsaturation of the substrate with the liquid phase.

The preferred growing containers are multi-cavity nursery containers,commonly referred to as miniplug trays, flats, or cell-packs. Suchcontainers are commonly used to produce plant plugs that can bemechanically transplanted into larger containers for continued growthunder greenhouse conditions or into bare-root seedling beds in afield-growing environment. The present invention can be practised byusing any multi-cavity trays, or alternatively, with individual pots. Anon-limiting example are miniplug trays filled with 100% short-fibrepeat product polymerised with a water-binding polymer.

Nursery containers sown with conifer somatic embryos or germinants arepreferentially placed in a conventional plant propagation environment.The desired plant propagation environment consists of, but is notlimited to, the ranges of temperatures of 15-35° C., relative humiditiesof 50-100%, photosynthetic photon flux of 15-500 μmol m⁻²s⁻¹ PAR,diurnal cycles of 6 h day/18 h night to 22 h day/2 h night, and a carbondioxide concentration of 0.003-0.02%. Although other conditions mayvary, it is preferable that atmospheric relative humidity around thecontainers sown with conifer somatic embryos is maintained in the rangeof 80-100 % for at least the first three to seven days. There are manymethods that may be used to achieve these levels of relative humidity. Anon-limiting example is to place the containers in a greenhouseenvironment with misting or fogging equipment that is deployed atcontrolled intervals. Another non-limiting example is to place thecontainers in a fogging or misting tent, chamber or room such as, butnot restricted to, a horticulture chamber or room. After the germinantsare established as evidenced by the growth or furthered development ofcotyledons, epicotyl and root structures, the atmospheric relativehumidity may be gradually reduced and the germinants integrated intoconventional nursery cultural practices.

Micro-organisms such as insects, fungi, bacteria, yeast, and algae areubiquitous in conventional plant propagation substrates, equipment,containers and growing environments. Because the present invention ispractised in non-sterilised conditions, this presence in the environmentmay reach a pathogenic level at various stages of plant development. Itis therefore prudent that normal nursery pest management and hygienemeasures are practised to reduce the probabilities of an outbreak. Whenpests do occur, a wide variety of chemicals and biological pesticideproducts are available to control and eradicate plant pathogens.Pesticides such as Gnatrol®, Benlate®, Rovril®, Trumpet®, which areregistered for pest control in plant crops, can be used in conjunctionwith somatic embryos or germinants. A non-limiting prevention example isto incorporate Benlate (0.1 g/l) and Ampicillin (0.1 g/l) in thenutrient medium so that the immediate rooting zone environment is keptfree of fungi and bacteria for as long as the pesticides are still ineffect. Another non-limiting example is to use 5 -10 ml Gnatrol®/l toeradicate fungus gnat larvae.

As already noted above, the solid component contained in the nutrientmedium provides continuing physical support for the embryo or germinant,even after the liquid or semi-solid components have dispersed. If asolid component of this kind is not provided, the embryo or germinantmay begin to develop a rudimentary root, but the root may beinsufficient to anchor it in the growing substrate before thesurrounding nutrient medium disperses. The embryo or germinant mayconsequently lose its desired orientation with respect to the surface ofthe growing substrate and may consequently face in the wrong directionor loose contact between the root and the growing substrate. In eithercase, conversion to healthy plants will be compromised. In contrast, thesolid component becomes packed around the embryo or germinant and keepsit in the correct orientation. Any amount of solid in the nutrientmedium will help with this objective, but clearly more is better thanless provided there is not so much that the fluidity of the medium andits nutrient and water contents are compromised. Suitable ranges ofamounts can be determined experimentally, but the indications providedabove are effective.

The support of the embryo or germinant can be further enhanced byproviding a depression for the embryo or germinant and nutrient mediumin the surface of the growing substrate. This is illustrated in FIGS. 1to 5 of the accompanying drawings. FIG. 1 shows a nursery container 10,preferably a mini-plug tray (the container 10 forming one well of theminiplug tray) filled almost to the top with a porous solid growthsubstrate 11, e.g. soil, peat or a soil-like product bound together witha polymer. A slight depression 12 is made in the upper surface 13 of thesubstrate, the inside of the depression has a substrate surface 14.

As shown in FIG. 2, a flowable or semi-solid nutrient medium 15 isdispensed onto the substrate 11 in the container 10 from a hollowtubular device 16 having a lowermost orifice 17. The medium has afluidity such that it can be dispensed in this way by gravity or bypressure (e.g. elevated air pressure). The medium 15 enters thedepression 12 and contacts the inner substrate surface 14 and forms apool or body 18 on the upper surface 13.

As shown in FIG. 3, a somatic embryo (or germinant) 20 is then sown inthe container 10. This is achieved by a holding device (e.g. a tubeconnected to a vacuum pump—not shown in the drawing) which brings theembryo (or germinant) 20 into contact with the pool or body 18 ofnutrient medium 15. The embryo (or germinant) does not have to besubmerged in the nutrient medium and may be only partially submerged asshown in FIG. 3 with at least part of the embryo exposed to air.However, the medium provides support for the embryo (or germinant) sothat it remains in the desired upright orientation.

The pool or body 18 of nutrient medium 15 has sufficient cohesiveness toremain in place in contact with the embryo (or germinant) at least untilgermination of the embryo is complete, thus assuring the embryo (orgerminant) a source of water and carbohydrate nutrient. Eventually,though, the flowable or semi-solid component of the nutrient mediumdisperses, e.g. by being absorbed into the growth substrate 11 or bybeing broken down by microorganisms. When this takes place, the solidcomponent 25 of the nutrient medium remains in place around the embryo(or germinant) 20 as shown in FIG. 4. This provides the embryo (orgerminant) with physical support to prevent it toppling over orotherwise being dislodged while the emergent root 26 is still in ajuvenile condition. This keeps the embryo (or germinant) facing in thecorrect upright direction until the root becomes established, as shownat 26′, and the seedling 28 becomes developed (FIG. 5). The solidcomponent of the nutrient medium may become an indistinguishable part ofthe growth substrate as the seedling develops.

The following Examples are provided to further illustrate the presentinvention and are not to be construed as limiting the invention in anymanner.

EXAMPLE 1

The objective of this experiment was to study the effects of differentcarbohydrate compositions in nutrient medium used for ex vitro growth ofconifer germinants from somatic embryos.

Methods

Traditionally, sucrose is the primary carbon and energy source in planttissue culture media. Mature plant somatic embryos are routinelygerminated in vitro on sucrose nutrient medium and grown intotransplantable plants. Maltose and other soluble carbohydrates may beused in various stages of somatic embryogenesis, such as in maintenancemedia for tissue proliferation and in maturation media for somaticembryo induction (see U.S. Pat. Nos. 4,801,545, 5,036,007 and5,563,061). In in vitro studies carried out by the inventors of thepresent invention have shown that a variety of carbon sources, includinga combination of glucose and fructose as well as sucrose, can promotegrowth of loblolly pine somatic embryos. Since sucrose hydrolyses intoglucose and fructose in the medium (Tremblay and Tremblay, 1995), it ishypothesized that mixtures of simple carbohydrates, as compared tomonotype carbohydrate, may similarly promote or improve growth ofconifer somatic germinants.

Somatic embryos of Interior spruce (genotype IS 1) were desiccated andfrozen-stored for 8 months. Somatic embryos of loblolly pine (genotypeLP 1) and radiata pine (genotype RP 1) were stored in high relativehumidity treatment (HRHT) plates at 4° C. for 9 months (according toRoberts U.S. Pat. No. 5,183,757). Somatic embryos of Douglas-fir(genotype DF 1) were desiccated and stored at 4° C. for 4 months. Inthis experiment, somatic embryos of interior spruce were first culturedon a 0.8% w/v phytagel semi-solid medium at room temperatures for twodays. The cultured embryos, together with loblolly pine and radiata pineembryos, were then transferred to liquid medium (according to Fan andJanic U.S. patent application Ser. No. 09/550,110) for four days undersimilar conditions to complete pre-germination. This involved placing 80embryos (for spruce, embryos cultured first on semi-solid medium) fromeach species in 50 ml of medium with 3% w/v sucrose, ½ m24GMD nutrientsolution, 0.1 μM IBA, 0.6 mg/l L-glutamine, 0.05 mg/l L-alanine, 0.02mg/l L-cysteine-HCl, 0.6 mg/l L-arginine, 0.01 mg/l L-leucine, 2.0 mg/lglycine, 2.0 mg/l serine, 0.5 mg/l L-proline, and 100,000 units/lpenicillin, in a 250 ml Kimax® baffled Erlenmeyer culture flask. Theflasks were place on a gyratory shaker (60 rpm) at ambient temperatures(20-23° C.) under a day/night diurnal cycle of 18/6 hours at 20-30 μmolm⁻²s⁻¹ PAR provided by two 20-W cool white fluorescent lights.Douglas-fir embryos were first cultured on the same semi-solid medium at12° C. for one week before being transferred to liquid medium for oneday as described above. The liquid medium for Douglas-fir embryos alsocontained 0.1 g/l Ampicillin and 0.1 g/l Benlate.

Germinants were sown ex vitro in miniplug trays. Each cell in theminiplug trays was filled with 0.3 ml of nutrient medium in the centerof the polymer-bound peat growing-medium. The nutrient medium consistedof the following basic ingredients: 0.2% w/v phytagel, 4% w/vα-cellulose, ½m24GMD, 0.1 μM IBA, 0.1 μM BA, 0.1 g/l Benlate, and 0.1g/l Ampicillin. Carbohydrate compositions and concentrations in thenutrient medium were as described in Table 1.1. Note that the mediumvolume was adjusted to accommodate the fact that 1 g of alpha celluloseoccupied 1 ml of medium. In order to avoid variation in osmoticpotential in the nutrient medium, the total molar concentration ofcarbohydrates in each treatment was fixed at the same level (i.e. 116.9mM). Each treatment had 4 replicates of 21 germinants per genotype. Theroot radicles of the pre-germinated embryos were inserted by hand intothe nutrient medium. After sowing, the miniplug trays were placed in ahorticultural germination chamber with conditions, and fertility andpesticide regime similar to those described in Example 2. The survivalpercentage and morphological development results of the germinants weredetermined after three weeks of growth under non-sterile ex vitroconditions.

Results

The three-week ex vitro results are summarized in Table 1.1. Nosignificant differences in %-survival and germinant growth were foundamong the treatments of nutrient medium with different carbohydratesources tested.

Sucrose is the traditional carbohydrate used in tissue culture media.This example illustrated that the growth of somatic germinants fromdifferent conifer species can similarly benefit from different types ordifferent combinations of carbohydrates in the nutrient medium. Avariety of carbohydrate compositions can be used in nutrient medium toenhance germinant performance for each of the commercially importantconifer species tested. TABLE 1.1 Sucrose, glucose, fructose, andmaltose concentrations in the various nutrient media and three-weeksurvival percentage, and morphological development of conifer somaticembryos under ex vitro non-sterile conditions Carbohydrate composition(g/l) Shoot Root SPECIES Genotype Sucr. Gluc. Fruct. Malt. Survival (%)length (mm) length (mm) 40 0 0 0 97.6 ± 1.4 16.6 ± 0.5 15.3 ± 1.6  20 00 21.05 91.7 ± 4.1 16.9 ± 0.6 19.1 ± 1.8  13.33 7.02 7.02 0 88.1 ± 7.116.4 ± 0.5 18.5 ± 2.0  13.95 12.24 1.80 0 86.9 ± 2.3 16.2 ± 0.6 17.7 ±1.9  INTERIOR IS 1 9.19 8.11 8.11 0 83.3 ± 7.9 19.7 ± 2.6 19.8 ± 1.8 SPRUCE 9.19 6.31 6.31 7.21 88.1 ± 7.5 16.7 ± 0.6 16.7 ± 1.9  0 7.21 3.6020.49 89.3 ± 4.5 15.3 ± 0.5 16.2 ± 1.8  0 10.53 10.53 0 91.7 ± 4.1 17.8± 0.6 19.5 ± 1.7  0 0 0 42.10 96.4 ± 1.2 14.4 ± 0.4 12.5 ± 1.5  40 0 0 094.0 ± 3.0  8.1 ± 0.3 3.1 ± 0.7 20 0 0 21.05 94.0 ± 3.0  8.0 ± 0.3 2.9 ±0.6 13.33 7.02 7.02 0 86.9 ± 2.3  8.5 ± 0.3 1.8 ± 0.3 13.95 12.24 1.80 0 71.4 ± 11.5  8.2 ± 0.4 3.0 ± 0.8 LOBLOLLY LP 1 9.19 8.11 8.11 0 85.7 ±4.8  7.6 ± 0.3 1.8 ± 0.4 PINE 9.19 6.31 6.31 7.21 81.0 ± 4.3  8.1 ± 0.42.7 ± 0.5 0 7.21 3.60 20.49 81.0 ± 5.1  8.1 ± 0.3 3.1 ± 0.5 0 10.5310.53 0 91.7 ± 3.0  9.0 ± 0.3 3.8 ± 0.7 0 0 0 42.10 82.1 ± 7.9  7.4 ±0.3 2.5 ± 0.6 40 0 0 0  63.1 ± 11.1  7.5 ± 0.3 1.5 ± 0.5 20 0 0 21.0564.3 ± 7.4  7.5 ± 0.3 3.0 ± 0.6 13.33 7.02 7.02 0 66.7 ± 4.3  7.7 ± 0.32.4 ± 0.6 13.95 12.24 1.80 0  53.6 ± 10.5  7.3 ± 0.3 2.0 ± 0.4 RADIATARP 1 9.19 8.11 8.11 0 73.8 ± 8.1  7.3 ± 0.3 2.4 ± 0.5 PINE 9.19 6.316.31 7.21  54.8 ± 14.1  7.3 ± 0.3 2.6 ± 0.6 0 7.21 3.60 20.49 58.3 ± 6.6 7.0 ± 0.3 1.9 ± 0.4 0 10.53 10.53 0 82.1 ± 6.3  7.9 ± 0.3 1.7 ± 0.2 0 00 42.10 70.2 ± 6.3  6.7 ± 0.3 2.1 ± 0.6 40 0 0 0 86.9 ± 2.3 12.1 ± 0.44.1 ± 0.5 DOUGLAS- DF 1 20 0 0 21.05 89.3 ± 3.0 11.4 ± 0.4 5.0 ± 0.6 FIR13.33 7.02 7.02 0  76.2 ± 10.1 12.4 ± 0.4 4.6 ± 0.5 13.95 12.24 1.80 064.3 ± 8.8 11.9 ± 0.4 4.8 ± 0.4 9.19 8.11 8.11 0 77.4 ± 7.9 11.2 ± 0.35.0 ± 0.9 9.19 6.31 6.31 7.21 79.8 ± 4.9 13.1 ± 0.5 5.8 ± 0.7 0 7.213.60 20.49 67.9 ± 9.0 11.5 ± 0.3 5.3 ± 0.5 0 10.53 10.53 0  69.0 ± 12.112.6 ± 0.5 6.2 ± 0.5 0 0 0 42.10 86.9 ± 6.0 11.3 ± 0.3 6.7 ± 0.7

EXAMPLE 2

The objective of this study was to compare the effect of differentgelling agents in nutrient medium on non-sterile ex vitro growth ofconifer germinants from somatic embryos.

Methods

Many gelling agents are currently being used in the production of tissueculture media. Agar is by far the most popular although phytagel isgaining recognition. Gelcarin is a cheaper alternative to agar andphytagel. This experiment compared the use of agar, phytagel, andgelcarin in the nutrient medium and assessed their effects on ex vitrogrowth of germinants from conifer somatic embryos.

Before experimental use, somatic embryos of interior spruce (genotypeIS 1) were desiccated and frozen-stored for seven months. Somaticembryos of radiata pine (genotype RP 2) were stored in HRHT plates@4° C.for ten and nine months, respectively. In the experiment, somaticembryos of interior spruce were first cultured on a 0.8% w/v phytagelsemi-solid medium at room temperatures for one day. These embryos, aswell as radiata pine embryos, were then cultured in liquid medium forfour days as described in Example 1.

Germinants were sown ex vitro in miniplug trays. Each cell in theminiplug trays contained 0.3 ml of nutrient medium. The nutrient mediumconsisted of the following basic ingredients: 4% w/v α-cellulose, 4% w/vsucrose, ½ m24GMD, 0.6 mg/l L-glutamine, 0.05 mg/l L-alanine, 0.02 mg/lL-cysteine-HCl, 0.6 mg/l L-arginine, 0.01 mg/l L-leucine, 2.0 mg/lglycine, 2.0 mg/l serine, 1.0 mg/l proline, 0.1 g/l Benlate, 0.1 g/lAmpicillin, 0.1 μM IBA and 0.1 μM BA. The gelling agents and theirconcentrations in the nutrient medium were described in Table 2.1. Notethat the medium volume was adjusted to accommodate the fact that 1 g ofalpha cellulose occupied 1 ml of medium. After sowing was completed, thetrays were placed in a horticultural growth chamber. Environmentalconditions in the chamber were as follows: 20-90 μmol m⁻²s⁻¹ PARprovided by eight 40-W fluorescent lights, 22-25° C. air temperature,and 60-100% RH. During the experimental period, 0.1 g/l Benlate, 0.1 g/lAmpicillin solutions were used to control fungi and bacteria whenneeded. On the first day of the third week, all treatments werefertilized with 0.5 g/l 20-20-20 all-purpose fertilizer. The %-survivaland morphological development of germinants were determined after threeweeks of growth under non-sterile ex vitro conditions.

Results

This example demonstrated that variation in the type and strength of thegel in the nutrient medium had no significant effect on the survival andmorphological development of the germinants after three weeks ofnon-sterile ex vitro growth (Table 2.1). Though gel strength and typehad no effect on germinant growth, the inclusion of a gelling agent inthe medium reduces the separation of liquid and solid phases of themedium. This helps to keep dissolved nutrients in a more confined regionof the miniplug, where the germinant is establishing early growth. Thepresence of a gelling agent in the medium also improves the structuralintegrity of the medium. Although this has no effect on germinantgrowth, it is important to help improve germinant stability inminiplugs. This is important to help prevent germinants from beingwashed out of miniplugs during watering and fertilization prior toextensive root development. It is especially important for germinantculture in a nursery (as opposed to chamber) environment (see Example10). In addition, gelling agents in nutrient medium may also help tokeep dissolved nutrients near the germinants for a longer period of timethan if the nutrients were dissolved in a fluid that could run down intothe subtending miniplug cells. TABLE 2.1 %-survival and morphologicaldevelopment of conifer germinants from somatic embryos after three weeksof growth under non-sterile ex vitro conditions Geno- Agar PhytagelGelcarin Shoot Root SPECIES type (% w/v) (% w/v) (% w/v) Survival (%)length (mm) length (mm) INTERIOR IS 1 0 0 0  86.8 ± 11.5 13.8 ± 0.6 4.4± 0.6 SPRUCE 0.3 95.0 ± 3.5 13.2 ± 0.5 4.3 ± 0.5 0.4 91.3 ± 2.4 11.0 ±0.5 4.7 ± 0.4 0.5 96.3 ± 2.4 12.4 ± 0.4 3.8 ± 0.5 0.1 92.5 ± 3.2 13.6 ±0.5 3.3 ± 0.5 0.2 90.0 ± 5.0 11.6 ± 0.3 3.1 ± 0.3 0.3 95.2 ± 3.4 11.8 ±0.5 3.6 ± 0.6 0.3 96.3 ± 1.3 13.6 ± 0.6 5.2 ± 0.7 0.4 88.8 ± 6.6 12.4 ±0.5 5.8 ± 0.7 0.5 81.3 ± 9.7 11.8 ± 0.4 4.2 ± 0.3 RADIATA RP 2 0 0 048.0 ± 6.3  8.0 ± 0.5 2.8 ± 0.4 PINE 0.3 68.8 ± 9.2 11.1 ± 1.5 2.6 ± 0.30.4 63.8 ± 8.3  8.6 ± 0.5 3.5 ± 0.3 0.5 81.3 ± 8.3  9.2 ± 0.4 1.4 ± 0.30.1 66.3 ± 9.0 11.2 ± 0.5 2.0 ± 0.5 0.2  77.5 ± 12.7  9.4 ± 0.5 1.8 ±0.2 0.3 76.5 ± 5.1 10.2 ± 0.5 2.1 ± 0.3 0.3 68.8 ± 6.3 10.2 ± 0.4 1.8 ±0.3 0.4  60.0 ± 10.8  9.5 ± 0.4 2.2 ± 0.3 0.5 75.0 ± 5.4 10.5 ± 0.6 3.4± 0.7

EXAMPLE 3

The objective of this study was to examine various concentrations ofα-cellulose in nutrient medium used for non-sterile ex vitro growth ofgerminants from conifer somatic embryos.

Methods

Experimental embryos were taken from interior spruce (genotype IS 2),stored on HRHT plates at 4° C. for six months), radiata pine (genotypeRP 3, stored in HRHT plates at 4° C. for six months) and Douglas-fir(genotypes DF 2 and 3, desiccated and stored @4° C. for two months).Embryos of interior spruce and radiata pine were pre-germinated for fourdays following the same protocol as described in Example 1. Douglas-firembryos were cultured on a semi-solid medium for two weeks @1 2° C.before receiving a one-day liquid culture treatment (also as describedin Example 1).

Germinants were sown ex vitro in miniplug trays as described inExample 1. To each cell in the miniplug trays, 0.3 ml of nutrient mediumwas added. The nutrient medium consisted of the following ingredients:0.2% w/v phytagel, 4% w/v sucrose, ½ m24GMD, 0.1 μM IBA, 0.6 mg/lL-glutamine, 0.05 mg/l L-alanine, 0.02 mg/l L-cysteine-HCl, 0.6 mg/lL-arginine, 0.01 mg/l L-leucine, 2.0 mg/l glycine, 2.0 mg/l serine, 0.1g/l Benlate, and 0.1 g/l Ampicillin. The α-cellulose fiber was tested at0, 3, 5, 7, and 10 % w/v levels. Each of these treatments had fourreplicates of 21 germinants sown for each Genotype. Note that the mediumvolume was adjusted to accommodate the fact that 1 g of alpha celluloseoccupied 1 ml of medium.

The root radicles of germinants were inserted by hand into the nutrientmedium. After sowing, the miniplug trays were placed in a horticulturalgermination chamber with conditions, and fertility and pesticide regimessimilar to those described in Example 2. On the first day of the secondweek, a 0.3 ml liquid medium similar to the nutrient medium (withphytagel and α-cellulose absent) was added into each cell in eachnutrient medium treatment. The %-survival and morphological developmentof the germinants were determined after three weeks.

The reason for inclusion of α-cellulose into the nutrient medium is thatgelled nutrient medium sinks into miniplugs over a two-week period,which renders young germinants sown in miniplug cavities unstable. Inthis experiment, the inclusion of α-cellulose into the nutrient mediumwas intended to anchor the germinants and prevent toppling. Theα-cellulose had no intended nutritional benefit; however, this materialwas intended to provide structural support in the miniplug cavity. Thus,a neutral or positive effect on germinant performance was seen as abeneficial result for inclusion of α-cellulose into nutrient medium.

Results

For all species and genotypes tested, ex vitro %-survival was similarfor all nutrient medium treatments (Table 3.1). In addition, there wereno significant differences in shoot and root growth for all genotypes ofDouglas-fir, interior spruce and radiata pine tested in the controlnutrient medium treatment (0% α-cellulose) and the nutrient mediumtreatments containing α-cellulose.

The addition of α-cellulose in nutrient medium had neither a positivenor a negative effect on survival of germinants from conifer somaticembryos. However, the addition of α-cellulose to nutrient mediumprovided structural support to germinants and prevented toppling in theminiplug cavities. An α-cellulose level<3% w/v did not providesufficient support to germinants. Too much α-cellulose in nutrientmedium (>7% w/v) reduced the fluidity of the medium, making it difficultto apply the nutrient medium to miniplug trays. The use of α-cellulosebetween 3% w/v and 7% w/v α-cellulose concentrations consistentlyproduced a dispensable medium that provided sufficient structuralsupport to germinants, and did not sink into miniplugs during the threeweeks miniplugs were maintained in the horticultural growth chamber.TABLE 3.1 %-Survival and morphological development of Douglas-fir,interior spruce, and radiata pine somatic embryos under non-sterile exvitro conditions in response to GEM without α-cellulose and withα-cellulose at four concentrations. Geno- α-Cellulose Shoot Root Speciestype (% w/v) Survival (%) length (mm) length (mm) Douglas-fir DF 2 078.6 ± 5.7 13.5 ± 0.4 10.0 ± 0.5  3  69.0 ± 10.4 12.4 ± 0.4 8.6 ± 0.5 583.3 ± 4.1 13.8 ± 0.4 8.8 ± 0.4 7  72.6 ± 13.0 13.9 ± 0.5 10.0 ± 0.6  1083.3 ± 4.1 13.6 ± 0.4 9.7 ± 0.6 DF 3 0 90.5 ± 3.9 10.5 ± 0.3 5.1 ± 0.3 386.9 ± 4.9 10.2 ± 0.4 4.4 ± 0.3 5 94.0 ± 3.6 11.6 ± 0.4 4.9 ± 0.4 7 95.2± 3.4 10.9 ± 0.4 5.2 ± 0.3 10 96.4 ± 3.6 11.0 ± 0.3 5.4 ± 0.4 InteriorIS 2 0 81.0 ± 5.1 12.9 ± 0.4 6.4 ± 0.5 Spruce 3 92.9 ± 4.1 12.5 ± 0.47.1 ± 0.5 5 85.7 ± 2.7 12.5 ± 0.4 6.8 ± 0.7 7 95.2 ± 3.4 12.4 ± 0.4 6.4± 0.6 10 83.3 ± 3.1 11.9 ± 0.4 6.9 ± 0.6 Radiata RP 3 0 94.0 ± 2.3  4.0± 0.2 2.4 ± 0.1 Pine 3 95.2 ± 3.4  3.8 ± 0.2 2.1 ± 0.1 5 96.4 ± 2.3  4.3± 0.2 2.5 ± 0.3 7 91.7 ± 2.3  4.1 ± 0.2 2.2 ± 0.1 10 90.5 ± 3.9  3.7 ±0.2 2.1 ± 0.1

EXAMPLE 4

The objective of this study was to assess the ex vitro conversion ofinterior spruce and Douglas-fir germinants into fully functionalcommercial-grade seedlings.

Methods

Somatic embryos of interior spruce (genotypes IS 3, 4 and 5) were storedin HRHT plates for 13 months at 4° C. Embryos were pre-germinated forfour days in a liquid medium in vented Lifeguard© polycarbonate boxes ona gyratory shaker (85 rpm) in a growth chamber. The following conditionswere maintained in the growth chamber: 20/22° C. (cycling 16 and 8 hrsrespectively) with a one 1 h photoperiod of 10 μmol m⁻²s⁻¹ PAR. Thepre-germination solution contained 3% w/v sucrose, m24GMD or re-modifiedm24GMD, 0.1 μM IBA, and amino acids (as in Example 1).

Somatic embryos of Douglas-fir (genotypes 2a and 2b) were produced inbioreactors. Mature embryos were bulk-sorted and desiccated. Thedesiccated embryos were stored at 4° C. for 3 months. Embryos werecultured on semi-solid medium in petri plates for 18 days at 12° C.These embryos with normal morphology (i.e. with symmetrical cotyledons,a hypocotyl, and root meristem) were hand-selected from the populationand transferred to liquid culture for three days in 250 ml Kimax®baffled culture flasks. The flasks contained liquid medium made withm24GMD and 3% w/v sucrose, 0.1 μM IBA, and amino acids (as in Example1). The flasks were placed on a gyratory shaker (100 rpm) underlaboratory conditions of 19-22° C. ambient temperature and a one 1 hourphotoperiod of 10 μmol m⁻²s⁻¹ PAR provided by one 20-W fluorescentlight.

Germinants were sown ex vitro in miniplug trays containing nutrientmedium. The nutrient medium contained 0.2% w/v phytagel, 2% w/v sucrose,2% w/v maltose, 4.5% w/v cellulose, m24GMD or re-modified m24GMD, aminoacids, 0.1 μM IBA, 0.1 μM BA, 0.1 g/l Ampicillin, and 0.1 g/l Benlate.Note that the medium volume was adjusted to accommodate the fact that 1g of alpha cellulose occupied 1 ml of medium. For interior spruce, 8replicates of 20 germinants were sown for each genotype and liquidmedium treatment (i.e. m24GMD and rm24GMD). For Douglas-fir, onereplicate of 77 (genotype 2a) and one replicate of 83 (genotype 2b)germinants were sown.

Douglas-fir germinants were grown for three weeks, and interior sprucegerminants were grown for seven weeks in a horticultural growth chamber(under similar environmental conditions, and fertility and pesticideregimes as described in Example 2). Germinants were then transferred toa greenhouse environment. Douglas-fir was maintained for 13 weeks in thegreenhouse (for a total of 16 weeks growth ex vitro), and Interiorspruce was maintained for five weeks in the greenhouse (for a total of12 weeks ex vitro). Three environmental zones were required for growinggerminants into young somatic seedlings in a greenhouse environment.These three zones provided conditions that allowed for the transition ofgerminants from the chamber environment to the greenhouse environment(i.e., from low light levels & high humidity to decreasing humidity andhigher light levels). Listed below are the greenhouse environmentalconditions that the germinants were transitioned through during seedlinggrowth.

-   -   1) Temperature: from 22 to 16° C.    -   2) Lighting: from 100 to 1200 μmol m⁻²s⁻¹ PAR    -   3) Humidity: from 85-92% RH to 50-70% relative humidity    -   4) Fertilization/irrigation: from 11-41-8 NPK at 50 ppm N to        19-9-18 NPK at 100ppm N    -   5) Pest control—same as Example 2 throughout culture period        At the end of the ex vitro growth periods, %-survival,        %conversion and heights were measured for interior spruce and        Douglas-fir seedlings. In addition, merchantability was assessed        for Douglas-fir seedlings.

Results

The %-survival, %-conversion and heights of seedlings for threegenotypes of interior spruce are shown in Table 4.1, and forDouglas-fir, data are shown in Table 4.2. This example confirmed thatconifer somatic embryos can be germinated and then sown ex vitro inminiplugs with nutrient medium to produce merchantable miniplugseedlings (see also Example 8). TABLE 4.1 %-Survival, %-conversion, andheight of interior spruce somatic seedlings after twelve weeks of exvitro development. Re-m24GMD denotes re-modified m24GMD mediumtreatments. Genotype Medium Survival (%) Conversion (%) Height (mm) IS 3m24GMD 63.1 ± 4.6 58.8 ± 4.2 28.4 ± 1.1 Re-m24GMD 69.4 ± 4.8 58.8 ± 3.030.2 ± 3.4 IS 4 m24GMD 87.6 ± 1.9 84.8 ± 2.1 29.2 ± 0.7 Re-m24GMD 85.7 ±3.8 76.8 ± 3.3 29.7 ± 0.9 IS 5 m24GMD 23.1 ± 5.7 21.9 ± 5.3 14.0 ± 0.9Re-m24GMD 20.0 ± 2.1 18.1 ± 2.5 16.3 ± 1.1

TABLE 4.2 %-Survival, %-conversion, height, and merchantability ofminiplug seedlings produced from Douglas-fir germinants after 16 weeksof ex vitro development. Total Total Repli- Germinants SurvivalConversion Seedling % of Sown Seedling Genotype cate Sown (%) (%) GradeGerminants Height (mm) DF 2b 1 83 86.8 78.3 Merchant-able 71.1 37.8 ±1.3 Rejected 7.2 11.2 ± 1.2 DF 2a 2 77 87.0 68.8 Merchant-able 54.5 41.9± 1.5 Rejected 14.3 10.2 ± 1.1

EXAMPLE 5

The objective of this example was to determine a proper combination ofmethyl-cellulose and α-cellulose in nutrient medium in order to improvethe consistency of the medium as it relates to storage (especially toeliminate gel separation and clumping with medium storage) and retentionin the miniplug cavities.

Methods

Nutrient medium was prepared containing the following ingredients:rm8GMD macro nutrients, micro nutrients and vitamins, amino acids (Table5.1), 3% w/v sucrose, 0.1 μM IBA, 0.05 μM BA, Benlate (0.1 g/l),Ampicillin (0.1 g/l), alpha cellulose, methylcellulose and phytagel.TABLE 5.1 Amino Acids in Nutrient Medium. Amino acid mg/l L-Glutamine(mg/l) 73.00 L-Glutamic acid (mg/l) 36.75 L-Alanine (mg/l) 4.45L-Arginine (mg/l) 69.60 L-Leucine (mg/l) 6.55 Glycine (mg/l) 7.50L-Serine (mg/l) 10.50 L-Asparagine (mg/l) 33.00 L-Proline (mg/l) 23.0L-Histidine 7.76 L-Tryptophan 10.21

The phytagel, α-cellulose and methylcellulose were assessed at thelevels described in Table 5.2. Note that the medium volume was adjustedto accommodate the fact that 1 g of alpha cellulose occupied 1 ml ofmedium. Each of the nutrient medium combinations was placed in arefrigerator for one week to assess their stability (i.e., ability tostay as a mixture or to have separation of the liquid and solidcomponents). In addition, separate batches of these treatments wereapplied at 0.4 ml to 5 replicates of 20 miniplug cells, for eachtreatment, to assess their structural stability in the miniplug cells.TABLE 5.2 Treatments modifying nutrient medium consistency with varyingconcentrations of methylcellulose, phytagel and α-cellulose.Treatments 1. 4.5% w/v α-cellulose, no methyl cellulose, 0.2% w/vphytagel (SOP) 2. 4.5% w/v α-cellulose, 1% w/v methyl cellulose, 0.1%w/v phytagel 3. 5% w/v α-cellulose, 1% w/v methyl cellulose, 0.1% w/vphytagel 4. 4.5% w/v α-cellulose, 1% w/v methyl cellulose, 0.2% w/vphytagel 5. 5% w/v α-cellulose, 1% w/v methyl cellulose, 0.2% w/vphytagel 6. 4.5% w/v α-cellulose, 0.75% w/v methyl cellulose, 0.1% w/vphytagel 7. 5% w/v α-cellulose, 0.75% w/v methyl cellulose, 0.1% w/vphytagel

Results

Addition of 0.75% - 1.0% w/v. methylcellulose in combination with 0.1%-0.2% w/v phytagel plus 4-4.5% w/v α-cellulose improved the consistencyof nutrient medium (i.e. prevented gel separation and provided smoothtexture) (Table 5.3).

It is possible to store medium with methylcellulose for at least 4 to 7days without the occurrence of gel separation or clumping. The mediumshould be stored at 4° C. to prevent microbial growth. As antibiotics insolution do not remain stable for more than 3 days, it is recommended toadd antibiotics to medium just prior to use after the storage period.

The addition of 0.75% -1.0% w/v methylcellulose in combination with 0.1%-0.2% w/v phytagel and 4-4.5% w/v α-cellulose improved the consistencyof the nutrient medium in the miniplug cells. The addition ofmethylcellulose also allowed for storage for 4-7 days at 4° C. (ifheat-sensitive components were added just prior to use). It ispreferable to incorporate 0.8% w/v methylcellulose with 0.2% w/vphytagel +4.5% w/v α-cellulose in the nutrient medium. TABLE 5.3Evaluation of nutrient medium consistency Consistency with storage (4°C. Treatments and/or room temp.) Comments on consistency in theminiblock cells. 1. 4.5% w/v α-cellulose, Gel separates with Runny ascompared to media with methyl cellulose. no methyl cellulose overnightstorage and The liquid components with dissolved nutrients 0.2% w/vphytagel medium clumps and sugars seep out of α-cellulose matrix intoand through miniplugs. This causes the bottom of blocks to be stickywith nutrients and promotes microbial growth on the bottom of blocks. 2.4.5% w/v α-cellulose, Gel does not Dispenses well into the cells. 1% w/vmethyl cellulose, separate with storage Structure maintained over a oneweek period. 0.1% w/v phytagel and medium is smooth 3. 5% w/vα-cellulose, Gel does not Dispenses well into the cells. 1% w/v methylcellulose, separate with storage Structure maintained over a one weekperiod. 0.1% w/v phytagel and medium is smooth 4. 4.5% w/v α-cellulose,Gel does not Dispenses well into the cells. 1% w/v methyl cellulose,separate with storage Structure maintained over a one week period. 0.2%w/v phytagel and medium is smooth 5. 5% w/v α-cellulose, Gel does notDispenses well into the cells. 1% w/v methyl cellulose, separate withstorage Structure maintained over a one week period. 0.2% w/v phytageland medium is smooth 6. 4.5% w/v α-cellulose, Gel does not Dispenseswell into the cells. 0.75% w/v methyl cellulose, separate with storageStructure maintained over a one week period. 0.1% w/v phytagel andmedium is smooth Reduced amount of methyl cellulose maybe better forautomated dispensing 7. 5% w/v α-cellulose, Gel does not Dispenses wellinto the cells. 0.75% w/v methyl cellulose, separate with storageStructure maintained over a one week period. 0.1% w/v phytagel andmedium is smooth Reduced amount of methyl cellulose maybe better forautomated dispensing

EXAMPLE 6

The purpose of this experiment was to assess the performance of loblollypine germinants in nutrient medium containing a combination ofmethylcellulose and α-cellulose. In addition, this trial provides ameasure of conversion rates of germinants for twelve loblolly pinegenotypes grown ex vitro in nutrient medium.

Methods

Somatic embryos of loblolly pine (genotypes LP 2-13) were pre-germinatedusing conventional in vitro medium and culture conditions. Germinantswere then sown ex vitro into miniplugs containing 0.4 ml of nutrientmedium. The medium employed consisted of the following ingredients:rm8GMD macro nutrients, micro nutrients and vitamins, amino acids (seeExample 5), 3% w/v sucrose, 0.1 μM IBA, 0.05 μM BA, Benlate (0.1 g/l),Ampicillin (0.1 g/l), 4.5% w/v alpha cellulose, 0.8% w/v methylcelluloseand 0.2% w/v phytagel. Note that the medium volume was adjusted toaccommodate the fact that 1 g of alpha cellulose occupied 1 ml ofmedium. To sow germinants, the root radicle of the germinants wasinserted into the medium by hand. For each of the twelve genotypes,between 100 and 250 germinants were planted (in 2 to 5 replicates of 50germinants).

Prior to sowing and nutrient medium dispensing, miniblocks and rootingsponges were sanitized by applying hot water so that the rooting spongereached a temperature of 65° C. Miniblocks were then leached with plainwater to lower EC levels in blocks. The miniblocks were then fertilizedwith 11-41-8, NPK at 50 ppm nitrogen.

After sowing, the miniplug trays were placed in a greenhouse withsimilar environmental conditions, fertility and pesticide regimes asdescribed in Example 4, except germinants were not maintained inhorticultural chambers for any of the growth period. Instead, germinantswere only transitioned through greenhouse environments.

Conversion percentage and morphological development of germinants weredetermined after 12 weeks growth ex vitro. A converted seedling wasdefined as a seedling that had a shoot height of between 2.5 and 5.0 cmand had a root system that was well developed throughout the rootingsponge.

Results

The modified nutrient medium (i.e., modified to include 0.8% w/vmethylcellulose with 0.2% w/v phytagel and 4.5% w/v α-cellulose) wassuitable for converting young loblolly pine somatic germinants intoshippable miniplug seedlings. Nursery conversion rates ranged from 55%to 89% for individual genotypes (Table 6.1). The average conversion ratewas 67% for all genotypes. Trial results showed that somatic germinantsfrom a range of loblolly pine genotypes, could be planted into nutrientmedium, and grown under non-sterile nursery conditions to yieldcommercially viable conversion rates. TABLE 6.1 Conversion rate ofloblolly pine germinants into shippable miniplug seedlings when grownwith nutrient medium under standard (non-sterile) greenhouse conditionsGenotype Mean SE LP 2 55 5 LP 3 70 7 LP 4 57.5 9.5 LP 5 55 0 LP 6 70 10LP 7 89 5 LP 8 74 1 LP 9 68 0 LP 10 60 2 LP 11 72.5 2.5 LP 12 74 1 LP 1361 1 OVERALL: 67

EXAMPLE 7

The purpose of this example was to test the structural integrity ofnutrient medium in miniplugs, particularly when milled peat and/or othersoil mix components were used to replace α-cellulose in nutrient medium.

Methods

Standard nutrient medium consisted of the following ingredients: rm8GMDmacro nutrients, micro nutrients and vitamins, amino acids (see Example5), 3% w/v sucrose, 0.1 μM IBA, 0.05 μM BA, Benlate (0.1 g/l),Ampicillin (0.1 g/l), 0.8% w/v methylcellulose and 0.2% w/v phytagel.Apart from methylcellulose and phytagel, other structural componentsused (i.e. α-cellulose or peat and/or other soil mix components) aredescribed in Table 8.1. Non-heat sensitive components of nutrient mediumtreatments were autoclaved 15 to 20 minutes for sanitization. Heatsensitive ingredients (including vitamins B5 and B12, amino acids, IBA,BA, Benlate, Ampicillin, and methylcellulose) were added afterautoclaving. Note that the medium volume was adjusted to accommodate thefact that 1 g of alpha cellulose/1 g peat occupied 1 ml of medium.Medium volume was not adjusted to accommodate bentonite, vermiculite orperlite.

Media treatments (with structural components described in Table 7.1)were dispensed into separate 400-well miniplug blocks. One completeminiplug block was used per each treatment tested. Each miniplugreceived a 0.4 ml aliquot of medium treatment. Blocks were then placedin a greenhouse environment and subjected to a watering schedule inwhich blocks were watered once a day for 10 days and twice a day for 5days. This simulated a normal twelve-week watering schedule in agreenhouse environment where blocks are usually subjected to 20irrigations. Blocks were checked up to 3 times per week for 3 weeks todetermine whether certain media treatments were better able to maintainstructural integrity as compared to others (i.e. % plugs with sunkenmedia were recorded). TABLE 7.1 Nutrient medium treatments tested forstructural integrity. % % % % % α-cellulose Peat Vermiculite PerliteBentonite Treatment (w/v) (w/v) (w/v) (w/v) (w/v) 1 4.5 — — — —(control - SOP) 2 — 5 — — — 3 — 5 1 — — 4 — 5 — 1 — 5 — 5 — — 3 6 — 5 1— 1 7 5 0.5 — 2 8 — 5 — 1 1 9 — 5 — 0.5 2 10 — 5 0.5 0.5 1 11 — 4 1 1 2

Results

Standard nutrient medium containing 4.5% w/v α-cellulose: no sinking ofthe medium in 34% of the cavities (Table 7.2).

Nutrient medium containing a combination of 5% w/v peat and 3% w/vbentonite clay: no sinking of the medium in 98% of the cavities.

Nutrient medium containing any combination of peat and bentonite clay aspart of the mix: no sinking of the medium in >80% of the cavities.

Replacing α-cellulose with a combination of peat and bentonite clay inthe nutrient medium retained the structural integrity of nutrient mediumthroughout the time period tested, and supported seedlings grown in thenursery. TABLE 7.2 Response of treatments with nutrient medium to thevolume of water they would receive over a typical 12 week growingregime. %-Plugs with Sunken % Structural Integrity Material Plugs withSunken Gem after Period in Greenhouse (GH) nutrient medium Treat- α-Cel-Vermi- Per- Ben- No. of 3 days 5 days 8 days 10 days 12 days 15 days 16days % % Not ment lulose Peat culite lite tonite Plugs in GH in GH in GHin GH in GH in GH in GH Sunken Sunken 1 4.5 — — — — 400 21 30 51 122 181229 263 66 34 2 — 5 — — — 400 0 25 57 72 112 112 175 44 56 3 — 5 1 — —400 4 4 11 24 39 60 88 22 78 4 — 5 — 1 — 400 4 5 30 69 106 182 242 61 405 — 5 — — 3 400 0 0 0 0 0 7 9 2 98 6 — 5 1 — 1 400 2 2 20 20 26 59 16642 59 7 — 5 0.5 — 2 400 1 2 4 13 13 35 52 13 87 8 — 5 — 1 1 400 1 1 8 2022 76 88 22 78 9 — 5 — 0.5 2 400 3 4 12 42 57 71 71 18 82 10 — 5 0.5 0.51 400 4 4 23 61 94 123 169 42 58 11 — 4 1 1 2 400 0 0 2 4 6 28 41 11 89

EXAMPLE 8

The purpose of this example was to test the effects of using milled peatand/or other soil mix components to replace α-cellulose in nutrientmedium while noting the effects on medium stability in miniplugs. Inaddition, this study tested the effects of using milled peat and/orother soil mix components in nutrient medium on loblolly pine germinantgrowth ex vitro.

Methods

Desiccated, mature loblolly pine embryos (genotype LP 11) were culturedon semi-solid medium for 7 days and then cultured in a liquid medium for5 days using methods similar to those described in Example 1. At the endof priming, germinants were sown in miniplugs with 0.4 ml of nutrientmedium with different structural components (Table 8.1). A total of 200to 300 germinants were sown per each treatment (in 2 to 3 replicates of100 germinants).

The standard nutrient medium consisted of the following ingredients:rm8GMD macro nutrients, micro nutrients and vitamins, amino acids (seeExample 5), 3% w/v sucrose, 0.1 μM IBA, 0.05 μM BA, Benlate (0.1 g/l),Ampicillin (0.1 g/l), 0.8% w/v methylcellulose and 0.2% w/v phytagel.Apart from methylcellulose and phytagel, other structural componentsused (i.e. α-cellulose or peat and/or other soil mix components) aredescribed in Table 8.1. Note that the medium volume was adjusted toaccommodate the fact that 1 g of alpha cellulose/1 g peat occupied 1 mlof medium. Medium volume was not adjusted to accommodate bentonite,vermiculite or perlite. Non-heat sensitive components of nutrient mediumtreatments were autoclaved 15 to 20 minutes for sanitization. Heatsensitive ingredients (including vitamins B5 and B12, amino acids, IBA,BA, Benlate, Ampicillin, and methylcellulose) were added afterautoclaving.

Germinants were grown under the standard nursery cultural regime (undergreenhouse environmental conditions, and fertility and pesticide regimesdescribed in Example 4), except germinants were not placed in ahorticultural growth chamber for any period prior to growing germinantsin the greenhouse. Instead, germinants were only transitioned throughgreenhouse environments. During the experiment, germinant survival andconversion rates were recorded every two weeks starting from the 2ndweek of growth after sowing ex vitro per each treatment. Nutrient mediumstability of the different treatments was also observed. TABLE 8.1Treatments for Nutrient Medium % alpha % % Vermi- % % Ben- cellulosePeat culite Perlite tonite Treatment (w/v) (w/v) (w/v) (w/v) (w/v) 1 4.5— — — — (control - SOP) 2 — 5 — — 3 — 5 — — 1.5 4 5 — — 3 5 — 5 0.5 1 26 — 5 — 0.5 2 7 — 4 1 1 2

Results

Survival and conversion data at 12 weeks of growth ex vitro showed thatgerminants sown in nutrient medium with 5% w/v peat with 1.5% w/v or 3%w/v bentonite clay had higher mean survival and conversion than othertreatments (Table 8.2). Nutrient medium with 4% w/v peat, 1% w/vvermiculite, 1% w/v perlite and 2% w/v bentonite also had high survivaland conversion rates. These treatments also provided long lastingstability to germinants in the miniplugs. It was observed that miniplugswith nutrient medium where α-cellulose was replaced with peat and 1.5%to 5% w/v clay did not show nutrient medium sinking into miniplugsduring the 12-week culture period. Replacing α-cellulose with peat andother soil mix components can improve ex vitro germinant conversion aswell as germinant stability in miniplugs. Improved conversion rates mayresult in part to the loss of fewer germinants due to their instabilityin miniplugs. TABLE 8.2 %-Survival and %-conversion for germinants grownin nutrient medium with different structural components % α- % % % % %-%- cellulose Peat Vermiculite Perlite Bentonite Survival ± Conversion ±Treatment (w/v) (w/v) (w/v) (w/v) (w/v) SE SE 1 4.5 — — — — 55.0 ± 2.054.5 ± 2.5 (control - SOP) 2 — 5 — —  56.3 ± 25.2  56.3 ± 25.2 3 — 5 — —1.5 76.3 ± 5.5 75.3 ± 5.2 4 5 — — 3 77.7 ± 7.5 76.0 ± 7.8 5 — 5 0.5 1 2 51.3 ± 21.7  50.3 ± 21.2 6 — 5 — 0.5 2  47.5 ± 26.5  47.5 ± 26.5 7 — 41 1 2 75.5 ± 8.5 73.5 ± 7.5

EXAMPLE 9

Nutrient medium is required for survival and conversion of conifersomatic embryos that are imbibed on semi-solid medium, primed in aliquid medium (as described in Example 1), and then sown ex vitro underhigh relative humidity conditions. This study was carried out todetermine whether liquid-primed germinants could germinate at a relativehumidity as low as 70%. In addition, this experiment compared thesurvival and conversion of germinants sown in polymerized peat plugscontaining nutrient medium with and without sucrose or structuralsupport.

Methods

Fresh, mature loblolly pine embryos (genotype LP 14) were stored on highrelative humidity treatment (HRHT) plates and subsequently cultured onsemi-solid medium for 7 days and then cultured in a liquid medium for 5days using methods similar to those described in Example 1. At the endof the liquid culture step, germinants were sown in miniplugs with 0.4ml of different treatments of nutrient medium (Table 9.1). Fourreplicates of 50 germinants were sown for each treatment. The standardnutrient medium consisted of the following ingredients: rm8GMD macronutrients, micro nutrients and vitamins, amino acids (see Example 5), 3%w/v sucrose, 0.1 μM IBA, 0.05 μM BA, Benlate (0.1 g/l), Ampicillin (0.1g/l), 0.8% w/v methylcellulose, 4.5% w/v α-cellulose and 0.2% w/vphytagel. Note that the medium volume was adjusted to accommodate thefact that 1 g of alpha cellulose occupied 1 ml of medium.

After sowing, germinants were monitored every second week for survival.In addition, conversion was monitored every second week, starting fromthe 4^(th) week of ex vitro growth until 12 weeks of growth in thenursery. TABLE 9.1 Treatments Ex vitro nutrient Treatment mediumtreatment Relative Humidity 1 Medium with 3% 95% RH 2 weeks (lab (exvitro SOP w/v sucrose chamber), then trans- control) ferred toGreenhouse Zone 1* 2 Medium with 3% 90% RH (lab chamber) w/v sucrose 3Medium with 0% 90% RH (lab chamber) sucrose 4 Medium without phytagel,90% RH (lab chamber) α-cellulose or methylcellulose 5 Medium with 3% 80%RH (Greenhouse) w/v sucrose 6 Medium with 3% 70% RH (HGC chamber) w/vsucrose*Greenhouse Zone 1 Environmental Conditions: Temperature - 22 to 16° C.,Lighting - 150 to 300 μmol m⁻²s⁻¹ PAR, Humidity - normally 92 to 85% RH,Fertigation - 11-41-8 at 50 ppm N, Pest control - same as Example 2

Results

Conversion rates were highest where germinants were maintained in 90-95%RH. Germinant conversion rates declined as the relative humidity droppedto 80% RH. Germinants did not survive when maintained at 70% RH. FIG. 6of the accompanying drawings is a graph showing the conversion rates ofloblolly pine embryos that were exposed to a range of humidity valuesduring ex vitro culture.

Germinants did not survive under ex vitro growing conditions when theywere not supplied with sucrose in the nutrient media. Germinants did notsurvive under ex vitro growing conditions when not supplied withstructural support in nutrient medium (i.e. α-cellulose, methylcelluloseand phytagel). FIG. 7 is a graph showing the conversion rate of loblollypine embryos under ex vitro conditions that were sown in nutrient mediumwith and without sucrose or structural support.

1. A method of sowing a heterotrophic somatic plant embryo or germinantof a conifer species to facilitate growth of the embryo or germinantinto an autotrophic seedling, which method comprises the followingsteps: providing a nutrient medium comprising particles of a solidcomponent contained within a flowable component containing water and acarbohydrate nutrient for the embryo or germinant, said flowablecomponent being selected from the group consisting of a fluid and asemi-solid; dispensing a quantity of the nutrient medium onto a surfaceof a porous solid growth substrate for the somatic plant embryo orgerminant and contacting said plant embryo or germinant with saidnutrient medium; and exposing said embryo or germinant to environmentalconditions effective for growth into an autotrophic seedling; wherein atleast said dispensing, contacting and exposing steps are carried out exvitro in non-sterile conditions; and wherein said particles forming saidsolid component are adapted to remain in contact with said embryo orgerminant after said flowable component undergoes dissipation in saidenvironmental conditions, thereby providing continuing physical supportfor said embryo or germinant after said dissipation.
 2. The method ofclaim 1, which comprises employing, as said flowable component, amaterial having a viscosity that causes at least some of said flowablecomponent containing the carbohydrate nutrient to remain in contact withsaid embryo or germinant until said embryo or germinant commencesautotrophic growth under said environmental conditions.
 3. The method ofclaim 1, which comprises employing, as said nutrient medium, a mediumthat has a fluidity under said environmental conditions that enables thenutrient medium to be dispensed under gravity or pressure from anorifice onto said porous solid growth substrate.
 4. The method of claim1, wherein said somatic plant embryo or germinant is contacted with saidnutrient medium after said nutrient medium has been dispensed onto saidsurface of the porous growth substrate.
 5. The method of claim 1,wherein said somatic plant embryo or germinant is contacted with saidnutrient medium before said nutrient medium has been dispensed onto saidsurface of the porous growth substrate.
 6. The method of claim 1,wherein said somatic plant embryo or germinant is contacted with saidnutrient medium as said nutrient medium is dispensed onto said surfaceof the porous growth substrate.
 7. The method of claim 1, wherein saidsomatic plant embryo or germinant is contacted with said nutrient mediumin such a way that part of said somatic plant embryo or germinantremains uncoated with said nutrient medium.
 8. The method of claim 1,wherein said porous solid growth substrate is in the form of a bodyprovided with a depression formed in said surface, and wherein saidnutrient medium is dispensed into said depression.
 9. The method ofclaim 8, wherein said embryo or germinant is at least partially insertedinto said depression in contact with said nutrient medium.
 10. Themethod of claim 1, wherein said embryo or germinant, when contacted withsaid nutrient medium, is naked.
 11. The method of claim 1, wherein saidsolid component of said nutrient medium comprises generally equi-axialparticles.
 12. The method of claim 1, wherein said solid component ofsaid nutrient medium comprise elongated particles.
 13. The method ofclaim 12, wherein said elongated particles comprise flexible fibers. 14.The method of claim 13, wherein said fibers are made of alpha-cellulose.15. The method of claim 14, wherein said nutrient medium containsmethylcellulose, agar, agarose, phytagel, gellan gum or gelcarin eithersingly or in combinations of two or more of the aforementioned gellingagents.
 16. The method of claim 1, wherein said solid component of saidnutrient medium comprises milled or sifted peat moss, perlite,vermiculite, clay, diatomaceous earth, coir or silica either singly orin combinations of two or more of the aforementioned components.
 17. Themethod of claim 16, wherein said nutrient medium containsmethylcellulose, agar, agarose, phytagel, gellan gum or gelcarin eithersingly or in combinations of two or more of the aforementioned gellingagents.
 18. The method of claim 1, wherein said flowable component ofsaid nutrient medium is a fluid comprising a viscous liquid containingsaid carbohydrate nutrient in a dissolved form.
 19. The method of claim1, wherein said flowable component of said nutrient medium is asemi-solid comprising a flowable gel containing said carbohydrate indissolved form.
 20. The method of claim 1, wherein said nutrient mediumadditionally comprises at least one material selected from the groupconsisting of mineral compounds, vitamins, amino acids, plant growthregulators and pest control compounds.
 21. The method of claim 1,wherein said carbohydrate nutrient is a monosaccharide.
 22. The methodof claim 21, wherein said monosaccharide is selected from the groupconsisting of glucose and fructose.
 23. The method of claim 1, whereinsaid carbohydrate nutrient is an oligosaccharide.
 24. The method ofclaim 23, wherein said oligosaccharide is selected from the groupconsisting of sucrose, maltose, raffinose, and stachyose.
 25. The methodof claim 1, wherein said carbohydrate nutrient is a combination ofmonosaccharides, a combination of oligosaccharides or a combination ormonosaccharides with oligosaccharides.
 26. The method of claim of 25,wherein said monosaccharides are selected from the group consisting ofglucose and fructose, and said oligosaccharides are selected from thegroup consisting of sucrose, maltose, raffinose, and stachyose.
 27. Themethod of claim 1, wherein said carbohydrate nutrient is sucrose presentin the nutrient medium in an amount in the range of in the range 1 and10% (w/v).
 28. The method of claim 1, wherein said carbohydrate nutrientis maltose present in the nutrient solution in an amount in the range ofin the range 1 to 10% (w/v).
 29. The method of claim 1, wherein saidsolid component is biologically inert.
 30. The method of claim 1,wherein said embryo or germinants are from the family Pinaceae.
 31. Themethod of claim 1, wherein said embryo or germinants are of tree speciesselected from the group consisting of Loblolly pine, Radiata pine,spruce and Douglas fir.
 32. A method of growing an autotrophic seedlingfrom a somatic plant embryo or germinant of a conifer species, whichmethod comprises the following steps carried out ex. vitro innon-sterile conditions: providing a nutrient medium comprising particlesof a solid component present within a flowable component, said flowablecomponent being selected from the group consisting of an aqueous fluidand an aqueous semi-solid, containing a carbohydrate nutrient for theembryo or germinant; dispensing a quantity of the nutrient medium onto asurface of a porous solid growth substrate for the somatic plant embryoor germinant held in a container; contacting said plant embryo orgerminant with said nutrient medium; and subjecting said embryo orgerminant to environmental conditions to further growth and developmentinto an autotrophic conifer seedling; wherein said nutrient medium has acohesiveness such that at least some of said fluid or semi-solidcontaining the carbohydrate nutrient remains in contact with said embryoor germinant as said embryo or germinant proceeds to develop underenvironmental conditions effective for said development; and whereinsaid particles of the solid component are adapted to remain in contactwith said embryo or germinant after of said flowable or semi-solidmaterial dissipates under said environmental conditions, therebyproviding continuing physical support for said embryo after saiddissipation.
 33. A nutrient medium for a somatic plant embryo orgerminant of a conifer species, which medium comprises particles of asolid component present within an aqueous flowable component comprisinga flowable or semi-solid component containing a carbohydrate nutrientfor the embryo or germinant, wherein said nutrient medium has aviscosity such that, when contacted with said embryo or germinant, atleast some of said flowable or semi-solid component containing thecarbohydrate nutrient remains in contact with said embryo or germinantas said embryo or germinant proceeds to grow and convert to anautotrophic seedling under environmental conditions effective for saidconversion, and wherein said particles of the solid component of thenutrient medium remain in contact with said embryo or germinant aftersaid flowable or semi-solid material dissipates, thereby providingcontinuing physical support for said embryo or germinant after saiddissipation.
 34. The medium of claim 33, which comprises employing, assaid nutrient medium, a medium that has a fluidity under saidenvironmental conditions such that it may be dispensed under gravity orpressure from an orifice onto said porous solid growth substrate. 35.The medium of claim 33, wherein said solid particles comprise flexibleor elongated fibers.
 36. The medium of claim 35, wherein said fibers aremade of alpha-cellulose.
 37. The medium of claim 36, wherein said fibresare present in a concentration range up to 10% (w/v).
 38. The medium ofclaim 36, wherein said fibers are present in the range between 3 and 8%(w/v).
 39. The medium of claim 36, wherein said medium also containsmethyl-cellulose, agar, agarose, phytagel, gellan gum, or gelcarineither singly or in combinations of two or more of the aforementionedgelling agents.
 40. The medium of claim 39, wherein themethyl-cellulose, agar, agarose, phytagel, gellan gum or gelcarinconcentrations in the nutrient medium ranges between 0 and 6% w/v. 41.The medium of claim 33, wherein said solid component comprises milled orsifted peat moss, perlite, vermiculite, clay, diatomaceous earth, coir,or silica either singly or with 2 or more of the aforementioned solidcomponents combined.
 42. The medium of claim 41, wherein said solidcomponents are present, either singly ore with two or more of the solidcomponents used in combination, in a concentration range up to a totalof 0.5 to 10% w/v.
 43. The medium of claim 41, wherein said medium alsocontains methyl-cellulose, agar, agarose, phytagel, gellan gum, orgelcarin either singly or in combinations of two or more of theaforementioned gelling agents.
 44. The medium of claim 43, wherein themethyl-cellulose, agar, agarose, phytagel, gellan gum or gelcarinconcentrations in the nutrient medium ranges between 0 and 6% w/v.